Compounds and Their Use for Reducing Uric Acid Levels

ABSTRACT

Bifunctional compounds that increase uric acid excretion and reduce uric acid production, and monofunctional compounds that either increase uric acid excretion or reduce uric acid production. Methods of using these compounds for reducing uric acid levels in blood or serum, for treating disorders of uric acid metabolism, and for maintaining normal uric acid levels in blood or serum are also provided. Pharmaceutical compositions comprising the bifunctional and monofunctional compounds are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/928,623, filed on Jul. 14, 2020, which is a continuation of U.S.application Ser. No. 16/310,921, filed on Dec. 18, 2018, which is theNational Phase entry of International Application No. PCT/US2017/038522,filed on Jun. 21, 2017, which claims priority to U.S. Provisional Appln.Ser. No. 62/356,685, filed on Jun. 30, 2016, and to U.S. ProvisionalAppln. Ser. No. 62/358,669, filed on Jul. 6, 2016, the disclosures ofwhich are incorporated herein by reference in their entireties.

TECHNICAL FIELD

This invention relates to pharmaceutical compositions and methods forreducing uric acid in blood or serum of a subject employing bifunctionaland monofunctional compounds as active agents.

BACKGROUND

Gout afflicts more than 8 million U.S. subjects, and is associated withchronic elevation of uric acid (UA) in blood. The incidence of thiscondition has doubled in the past ten years. When UA exceeds solubilitylimits, it forms crystals that settle into joints and kidney, causingsevere pain, destructive arthritis, and kidney failure. Treatment forchronic gout entails extended—if not lifelong—therapy focused onreducing UA production or increasing its excretion. The standard-of-carefor initial therapy of gout is allopurinol, a drug that inhibitsxanthine oxidase (XO), a key production enzyme. Launched in 2009,Uloric® (febuxostat; Takeda), has similar activity as an XO inhibitorwith somewhat higher efficacy and improved safety. Xanthine oxidaseinhibitors are used as initial therapy in more than 90% of goutpatients; nonetheless, the therapeutic target is achieved in less thanone-third of patients, the drugs have multiple side effects, andhypersensitivity (especially to allopurinol) is common. Given that mostpatients do not actually respond, the continued use of ineffectivetreatment administered over many months in order to determine the lowpercentage of patients who might respond represents an important burdento patients as well as substantial costs to global healthcare systems,Moreover, the high proportion of failures causes many patients to becomenon-compliant with therapy and thus at increased risk for development ofchronic complications of gout, especially destructive arthritis andrenal insufficiency.

Since 2000, rapid advances in the biology of proteins known astransporters have presented an array of new drug targets. The enzymeURAT1 is a high capacity renal transporter that reabsorbs most of the UAthat is initially filtered into the urine from the blood by the kidney.Inhibitors of certain urate transporters may prevent such reabsorptionand thereby increase UA excretion. Several drugs are now known toinhibit URAT1, including benzbromarone (approved but withdrawn in the USby Sanofi in 2003), and lesinurad (Zurampic®, AstraZeneca), which wasapproved in the U.S. and EU in 2016.

These drugs are all mono-functional. That is, they inhibit only one ofthe two equilibrium paths that reduce the levels of UA in blood (i.e.,decreased production or increased excretion). Allopurinol is an exampleof a drug that decreases UA production by inhibiting xanthine oxidase,but it has no effect on renal excretion. As expected, allopurinol doesnot affect the activity of URAT1 or other renal urate transporters.Benzbromarone and lesinurad increase UA excretion (i.e., they promoteuricosuria) primarily via inhibition of URAT1, but these agents have noeffect on UA production, since they have no substantial effect onxanthine oxidase. Since xanthine oxidase inhibition is the principal,preferred, and primary 1^(st)-line form of treatment for hyperuricemia,agents that promote uricosuria are used second-line and are commonlyemployed only in combination with xanthine oxidase inhibitors ratherthan as single-agents.

Non-sedating 5-carboxanilide derivatives of barbiturates, includingmerbarone (5-(N-phenylcarboxamido)-2-thio-barbituric acid), have beenevaluated as potential cytotoxic anticancer drugs. Subsequently, it wasdiscovered that clinical treatment with merbarone was associated with amarked reduction of UA levels in blood. Despite these discoveries, thecytotoxic activity of merbarone completely precluded its use as atreatment for a chronic lifelong disorder of UA metabolism, since thesafety of such use (primarily its genotoxicity) posed a serious risk toother aspects of human health. Such clinical utility would only bepossible if the genotoxic activity could be chemically dissociated andeliminated from the hypouricemic activity. The inventors have sincedescribed a number of non-genotoxic hypouricemic derivatives ofmerbarone.

There exists a compelling need for new drugs than can reduce UA levelsin blood and provide better treatment for patients afflicted by gout.Reduction in UA is universally acknowledged as beneficial for patientswith gout and other hyperuricemic disorders, and such reduction isdirectly linked to patient benefit. Reduced serum UA is accepted byinternational drug regulatory agencies (e.g., the U.S. Food and DrugAdministration [FDA], the European Medicines Agency [EMA], etc.) as anendpoint for commercial drug approval in these diseases. As previouslynoted, drugs that can overcome the limited clinical activity of xanthineoxidase inhibitors are available or are currently being investigated,but only as “add-ons” for combination use. The approval of lesinurad[Zurampic®] is the most recent example. The present invention relates tonew compounds that can provide alternatives to current therapy forelevated UA levels and treatment of disorders of UA metabolism such asgout. Certain of these compounds have the particular advantage ofbifunctional activity (i.e., decreasing UA production by inhibitingxanthine oxidase and increasing UA excretion by inhibiting a renal uratetransporter), making them suitable for use as initial therapy and assingle agents rather than “add-on” therapies. In addition, certain ofthe compounds have reduced toxicity compared to prior art drugs such asmerbarone.

SUMMARY

In a first aspect, compounds having a structure represented by Formula(I) are provided:

wherein

-   -   W, X, and Y are each independently O, S, NR² or N(R²)₂;    -   T is —CONR²—, —C(NR²)NH—, —C(NOR²)NH—, —C(N—NR²)NH—, —C(SR²)N—,        or —NHC(O)—;    -   A is phenyl, heteroaryl, C5-C10 branched or unbranched        cycloalkyl, C6-C10 bicycloalkyl or C5-C10 spirocycloalkyl;    -   each Z is independently present or absent and, if present, is        independently selected from one or more halogen atoms, —CN,        —CF₃, —OR², —C(O)R², SR², —S(O)_(g)R³ where g is 1 or 2,        —N(R²)₂, —NO₂, —CO₂R², —OCO₂R³, OC(O)R², —CON(R²)₂, —NR²C(O)R²,        —SO₂N(R²)₂, —NR₂SO₂R³, —NR²SO₂N(R²)₂ or —NR²C(O)N(R²)₂,        —C(O)NHOR², alkyl, aryl, alkenyl and alkynyl;        wherein each R² is independently H, alkyl or aryl;        wherein each R³ is independently alkyl or aryl, optionally        substituted with one or more halogen atoms or OR²; and        wherein a, b, c, d, and e are each independently carbon or        nitrogen, or four of a, b, c, d, and e are each independently        carbon or nitrogen and one of a, b, c, d, and e is O, with the        proviso that at least one of a, b, c, d and e is nitrogen, and Z        is not connected directly to nitrogen or oxygen.

In a second aspect, compounds having a structure represented by Formula(II) are provided:

wherein

-   -   W, X, and Y are each independently O, S, NR² or N(R²)₂;    -   A is phenyl, heteroaryl, C5-C10 branched or unbranched        cycloalkyl, C6-C10 bicycloalkyl or C5-C10 spirocycloalkyl;    -   each Z is independently present or absent and, if present, is        independently selected from one or more halogen atoms, —CN,        —CF₃, —OR², —C(O)R², SR², —S(O)_(g)R³ where g is 1 or 2,        —N(R²)₂, —NO₂, —CO₂R², —OCO₂R³, OC(O)R², —CON(R²)₂, —NR²C(O)R²,        —SO₂N(R²)₂, —NR²SO₂R³, —NR²SO₂N(R²)₂ or —NR²C(O)N(R²)₂,        —C(O)NHOR², alkyl, aryl, alkenyl and alkynyl;        wherein each R¹ is C1-C8 branched or unbranched alkyl,        optionally substituted with Z;        wherein each R² is independently H, alkyl or aryl;        wherein each R³ is independently alkyl or aryl, optionally        substituted with one or more halogen atoms or OR²; and        wherein a, b, c, d, and e are each independently carbon or        nitrogen, or four of a, b, c, d, and e are each independently        carbon or nitrogen and one of a, b, c, d, and e is O, with the        proviso that at least one of a, b, c, d and e is nitrogen, and Z        is not connected directly to nitrogen or oxygen.

In a third aspect, compounds having a structure represented by Formula(III) are provided:

wherein

-   -   W, X, and Y are each independently O, S, NR² or N(R²)₂;    -   each R² is independently H, alkyl or aryl; and    -   f is divalent —CR²—, —C(O)—, —SR², —S(O)_(g)— where g is 1 or 2,        —N(R′)₂—; or —C(—O(CR²)_(n)O—)— where n=2-3.

In a fourth aspect, compounds having a structure represented by Formula(IV) are provided:

wherein

-   -   W, X, and Y are each independently O, S, NR² or N(R²)₂;    -   each R² is independently H, alkyl or aryl; and    -   f is divalent —CR²—, —C(O)—, —S(O)_(g)— where g is 1 or 2,        —NR²—; or —C(—O(CR²)_(n)O—)— where n is 2-3.

In a fifth aspect, compounds having a structure represented by Formula(V) are provided:

wherein

W, X, and Y are each independently O, S, NR² or N(R²)₂; and

each R² is independently H, alkyl or aryl.

In a sixth aspect, compounds having a structure represented by Formula(VI) are provided:

wherein

-   -   W and X are each independently O, S, NR² or N(R²)₂;    -   A is phenyl, heteroaryl, C5-C10 branched or unbranched        cycloalkyl, C6-C10 bicycloalkyl or C5-C10 spirocycloalkyl;    -   each Z is independently present or absent and, if present, is        independently selected from one or more halogen atoms, —CN,        —CF₃, —OR², —C(O)R², SR², —S(O)_(g)R³ where g is 1 or 2,        —N(R²)₂, —NO₂, —CO₂R², —OCO₂R³, OC(O)R², —CON(R²)₂, —NR²C(O)R²,        —SO₂N(R²)₂, —NR²SO₂R³, —NR²SO₂N(R²)₂ or —NR²C(O)N(R²)₂,        —C(O)NHOR², alkyl, aryl, alkenyl and alkynyl;    -   U is —O—, —S—, —NR²— or —S(O)_(g)— where g is 1 or 2;        wherein each R² is independently H, alkyl or aryl;        wherein each R³ is independently alkyl or aryl, optionally        substituted with one or more halogen atoms or OR²; and        wherein a, b, c, d, and e are each independently carbon or        nitrogen, or four of a, b, c, d, and e are each independently        carbon or nitrogen and one of a, b, c, d, and e is O, with the        proviso that at least one of a, b, c, d and e is nitrogen, and Z        is not connected directly to nitrogen or oxygen.

In a seventh aspect, compounds having a structure represented by Formula(VII) are provided:

wherein

-   -   X and Y are each independently O, S, NR² or N(R²)₂;    -   Z is present or absent and, if present, is selected from one or        more halogen atoms, —CN, —CF₃, —OR², —C(O)R², SR², —S(O)_(g)R³        where g is 1 or 2, —N(R²)₂, —NO₂, —CO₂R², —OCO₂R³, OC(O)R²,        —CON(R²)₂, —NR²C(O)R², —SO₂N(R²)₂, —NR²SO₂R³, —NR²SO₂N(R²)₂ or        —NR²C(O)N(R²)₂, —C(O)NHOR², alkyl, aryl, alkenyl and alkynyl,        wherein each R² is independently H, alkyl or aryl;        wherein each R³ is independently alkyl or aryl, optionally        substituted with one or more halogen atoms or OR²; and        wherein a, b, c, d, and e are each independently carbon or        nitrogen, or four of a, b, c, d, and e are each independently        carbon or nitrogen and one of a, b, c, d, and e is O, with the        proviso that at least one of a, b, c, d and e is nitrogen, and Z        is not connected directly to nitrogen or oxygen.

In an eighth aspect, compounds having a structure represented by Formula(VIII) are provided:

wherein

-   -   W, X, and Y are each independently O, S, NR² or N(R²)₂;    -   T is —CONR²—, —C(NR²)NH—, —C(NOR²)NH—, —C(N—NR²)NH—, —C(SR²)N—,        or —NHC(O)—;    -   A is phenyl, heteroaryl, C5-C10 branched or unbranched        cycloalkyl, C6-C10 bicycloalkyl or C5-C10 spirocycloalkyl;    -   each Z is independently present or absent and, if present, is        independently selected from one or more halogen atoms, —CN,        —CF₃, —OR², —C(O)R², SR², —S(O)_(g)R³ where g is 1 or 2,        —N(R²)₂, —NO₂, —CO₂R², —OCO₂R³, OC(O)R², —CON(R²)₂, —NR²C(O)R²,        —SO₂N(R²)₂, —NR²SO₂R³, —NR²SO₂N(R²)₂ or —NR²C(O)N(R²)₂,        —C(O)NHOR², alkyl, aryl, alkenyl and alkynyl;        wherein each R² is independently H, alkyl or aryl;        wherein each R³ is independently alkyl or aryl, optionally        substituted with one or more halogen atoms or OR².

A further aspect relates to methods for reducing uric acid levels inblood or serum of a subject, or preventing elevation of uric acid levelsin blood or serum of a subject, comprising administering a compoundhaving a structure represented by Formula (I), Formula (II), Formula(III), Formula (IV), Formula (V), Formula (VI), Formula (VII), Formula(VIII), or a combination thereof, to a subject in need thereof in anamount effective to reduce blood or serum uric acid levels or preventelevation of blood or serum uric acid levels. In a modification of thisembodiment, the methods comprise administering a compound according to aspecific embodiment of the compounds of Formula (I), Formula (II),Formula (III), Formula (IV), Formula (V), Formula (VI), Formula (VII),Formula (VIII,) or a combination thereof, as described above, to asubject in need thereof in an amount effective to reduce blood or serumuric acid levels or prevent elevation of blood or serum uric acidlevels.

In certain embodiments of these methods, a compound having a structurerepresented by Formula (I), Formula (II), Formula (III), Formula (IV),Formula (V), Formula (VI), Formula (VII), Formula (VIII), or acombination thereof, is administered to a subject with gout,hyperuricemia, kidney disease, arthritis, kidney stones, kidney failure,urolithiasis, plumbism, hyperparathyroidism, psoriasis, inborn geneticerrors of metabolism (including but not limited to Lesch-Nyhansyndrome), sarcoidosis, cardiovascular disease (including but notlimited to atherosclerosis), or who is undergoing transplantation ofblood, bone marrow or solid organs to reduce uric acid levels.

A further aspect relates to methods for treating a disorder of uric acidmetabolism associated with or caused by elevated uric acid in blood orserum comprising administering to a subject in need thereof a compoundhaving a structure represented by Formula (I), Formula (II), Formula(III), Formula (IV), Formula (V), Formula (VI), Formula (VII), Formula(VIII), or a combination thereof, in an amount effective to reduce bloodor serum uric acid levels or prevent elevation of blood or serum uricacid levels, thereby treating the disorder of uric acid metabolism. Onesuch embodiment relates to methods for treating a disorder of uric acidmetabolism associated with or caused by elevated uric acid in blood orserum comprising administering to the subject a compound according to aspecific embodiment of the compounds of Formula (I), Formula (II),Formula (III), Formula (IV), Formula (V), Formula (VI), Formula (VII),Formula (VIII), or a combination, as described above.

A further aspect of the invention provides pharmaceutical compositionscomprising a compound having a structure represented by Formula (I),Formula (II), Formula (III), Formula (IV), Formula (V), Formula (VI),Formula (VII), Formula (VIII), or a combination thereof, and apharmaceutically acceptable carrier. In a specific embodiment, thepharmaceutical composition comprises a compound according to a specificembodiment of the compounds of Formula (I), Formula (II), Formula (III),Formula (IV), Formula (V), Formula (VI), Formula (VII), Formula (VIII),or a combination thereof, as described above.

A further aspect provides methods for synthesizing the compoundsdiscussed above, as discussed in more detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a general scheme for synthesis of compounds describedherein.

FIG. 2 illustrates a general scheme for synthesis of bridged compounds.

FIG. 3 illustrates an alternative general scheme for synthesis ofcompounds.

FIG. 4 illustrates a general scheme for synthesis of compoundscontaining a hydroxamic acid in place of the heterocyclic ring.

FIG. 5 illustrates a general scheme for synthesis of compoundscontaining a heterocycle fused onto the A ring.

FIG. 6 illustrates a general scheme for synthesis of compoundscontaining a substituent on X of the barbiturate ring.

FIG. 7 illustrates an alternative general scheme for synthesis ofcompounds containing a substituent on X of the barbiturate ring.

FIG. 8 illustrates a further alternative general scheme for synthesis ofcompounds containing a substituent on X of the barbiturate ring.

FIG. 9 illustrates an alternative general scheme for synthesis ofbridged compounds.

FIG. 10 illustrates a general scheme for synthesis of compoundscontaining a triazole heterocyclic ring.

FIG. 11 illustrates alternative general schemes for synthesis of the Aring of the compounds.

FIG. 12 illustrates a synthesis scheme for a compound having a structurerepresented by Formula (I_(a)), as described in Example 1.

FIG. 13 illustrates a synthesis scheme for a compound having a structurerepresented by Formula (I_(l)), as described in Example 2.

FIG. 14 illustrates a synthesis scheme for a compound having a structurerepresented by Formula (I_(b)), as described in Example 3.

FIG. 15 illustrates a synthesis scheme for a compound having a structurerepresented by Formula (I_(c)), as described in Example 4.

FIG. 16 illustrates a synthesis scheme for a compound having a structurerepresented by Formula (VI_(a)), as described in Example 5.

FIG. 17 illustrates a synthesis scheme for a compound having a structurerepresented by Formula (I_(d)), as described in Example 6.

FIG. 18 illustrates a synthesis scheme for a compound having a structurerepresented by Formula (I_(e)), as described in Example 7.

FIG. 19 illustrates a synthesis scheme for a compound having a structurerepresented by Formula (V_(a)), as described in Example 8.

FIG. 20 illustrates a synthesis scheme for a compound having a structurerepresented by Formula (VI_(b)), as described in Example 9.

FIG. 21 illustrates a synthesis scheme for a compound having a structurerepresented by Formula (IV_(a)), as described in Example 10.

FIG. 22 illustrates a synthesis scheme for a compound having a structurerepresented by Formula (VI_(c)), as described in Example 11.

FIG. 23 illustrates a synthesis scheme for a compound having a structurerepresented by Formula (I_(f)), as described in Example 12.

FIG. 24 illustrates a synthesis scheme for a compound having a structurerepresented by Formula (II_(a)), as described in Example 13.

FIG. 25 illustrates a synthesis scheme for a compound having a structurerepresented by Formula (I_(g)), as described in Example 14.

FIG. 26 illustrates a synthesis scheme for a compound having a structurerepresented by Formula (II_(b)), as described in Example 15.

FIG. 27 illustrates a synthesis scheme for a compound having a structurerepresented by Formula (VII_(a)), as described in Example 16.

FIG. 28 illustrates a synthesis scheme for a compound having a structurerepresented by Formula (I_(h)), as described in Example 17.

FIG. 29 illustrates a synthesis scheme for a compound having a structurerepresented by Formula (IV_(b)), as described in Example 18.

FIG. 30 illustrates a synthesis scheme for a compound having a structurerepresented by Formula (I_(m)), as described in Example 19.

FIG. 31 illustrates a synthesis scheme for a compound having a structurerepresented by Formula (III_(a)), as described in Example 20.

FIG. 32 illustrates a synthesis scheme for a compound having a structurerepresented by Formula (I_(n)), as described in Example 21.

FIG. 33 illustrates a synthesis scheme for a compound having a structurerepresented by Formula (I_(i)), as described in Example 22.

FIG. 34 illustrates a synthesis scheme for a compound having a structurerepresented by Formula (I_(j)), as described in Example 23.

FIG. 35 illustrates a synthesis scheme for a compound having a structurerepresented by Formula (I_(k)), as described in Example 24.

FIG. 36 illustrates a synthesis scheme for a compound having a structurerepresented by Formula (II_(c)), as described in Example 25.

FIG. 37 illustrates a synthesis scheme for a compound having a structurerepresented by Formula (VIII_(a)), as described in Example 26.

FIG. 38 illustrates a synthesis scheme for a compound having a structurerepresented by Formula (VIII_(b)), as described in Example 27.

FIG. 39 illustrates a synthesis scheme for a compound having a structurerepresented by Formula (IV_(c)), as described in Example 28.

FIG. 40 illustrates a synthesis scheme for a compound having a structurerepresented by Formula (II_(g)), as described in Example 29.

FIG. 41 illustrates a synthesis scheme for a compound having a structurerepresented by Formula (II_(d)), as described in Example 30.

FIG. 42 illustrates a synthesis scheme for a compound having a structurerepresented by Formula (II_(e)), as described in Example 31.

FIG. 43 illustrates a synthesis scheme for a compound having a structurerepresented by Formula (II_(h)), as described in Example 32.

FIG. 44 illustrates a synthesis scheme for a compound having a structurerepresented by Formula (II_(f)), as described in Example 33.

DETAILED DESCRIPTION

Before describing several exemplary embodiments provided herein, it isto be understood that the invention is not limited to the details ofconstruction or process steps set forth in the following description.The invention is capable of other embodiments and of being practiced orbeing carried out in various ways.

Reference throughout this specification to “one embodiment,” “certainembodiments,” “one or more embodiments” or “an embodiment” means that aparticular feature, structure, material, or characteristic described inconnection with the embodiment is included in at least one embodiment ofthe invention. Thus, the appearances of the phrases such as “in one ormore embodiments,” “in certain embodiments,” “in one embodiment” or “inan embodiment” in various places throughout this specification are notnecessarily referring to the same embodiment of the invention.Furthermore, the particular features, structures, materials, orcharacteristics may be combined in any suitable manner in one or moreembodiments.

As used herein, the term “bifunctional” with respect to disclosedcompounds means that the compound inhibits both a renal transporter,including but not limited to URAT1, and xanthine oxidase. The potency ofinhibition of either target may vary, but in general an IC50 of lessthan about 100 μM for both xanthine oxidase and a renal transporter suchas URAT1 is considered bifunctional. An IC50 of less than about 50 μMfor both xanthine oxidase and URAT1 is considered a particularly activebifunctional compound, and an IC50 of less than 10 μM is considered ahighly potent bifunctional compound.

As used herein, the term “monofunctional” with respect to disclosedcompounds means that the compound inhibits an enzyme in the uric acidmetabolic pathway involved in uric acid excretion that is either a renaltransporter, including but not limited to URAT1, or an enzyme involvedin uric acid production, including but not limited to xanthine oxidase,but not both. The potency of inhibition of single target may vary, butin general an IC50 of greater than about 100 μM for one of xanthineoxidase or URAT1, and an IC50 of less than about 100 μM for the other ofxanthine oxidase or URAT1, is considered monofunctional. An IC50 of lessthan about 50 μM for one of xanthine oxidase or URAT1, and an IC50 ofgreater than about 100 μM for the other of xanthine oxidase or URAT1, isconsidered a particularly active monofunctional compound. An TC50 ofless than about 10 μM for one of xanthine oxidase or URAT1, and an IC50of greater than about 100 μM for the other of xanthine oxidase or URAT1,is considered a highly potent monofunctional compound.

As used herein, the term “treatment” refers to reducing elevated uricacid levels in blood or serum, preferably by reducing levels to thenormal, low-normal or sub-normal range, with an overall goal ofrelieving symptoms and/or preventing recurrences of active disease. Forexample, a typical “therapeutic target” for treatment of elevated serumuric acid is a level ≤6.0 mg/dL. “Elevated” uric acid levels generallyrefer above-normal uric acid levels, as long-term elevated levels canresult in conditions that require additional treatment.

As used herein, the term “preventing” elevation of uric acid levels inblood or serum refers to maintaining normal or therapeuticallyacceptable uric acid levels in blood or serum in a subject who wouldotherwise experience an increase in uric acid levels, with an overallgoal of preventing development or recurrence of symptoms and/orpreventing recurrences of active disease. It will be appreciated thatprevention of elevation of uric acid levels is a goal of the long-termmaintenance therapy discussed below, as well as certain short-termconditions.

The numbering of the positions on the barbiturate ring used hereinfollows the convention of Warrell (U.S. Pat. No. 4,880,811). It is alsoto be understood that although the compounds disclosed herein aregenerally illustrated by specific chemical structures, the disclosure ofthe compounds is intended to include their tautomers. Representativeexamples of tautomers in the barbiturate ring include the structuresdepicted below, as well as any additional tautomers on the substituentsof Formula (I), Formula (II), Formula (III), Formula (IV), Formula (V),Formula (VI), Formula (VII), or Formula (VIII):

The compounds described herein meet certain needs in the therapeuticfield of reduction of uric acid levels in blood and treatment ofdisorders of uric acid metabolism that are associated with, or causedby, elevated uric acid levels in blood or serum. Certain of thecompounds are potent monofunctional inhibitors of URAT1 or xanthineoxidase. Certain of the compounds are bifunctional inhibitors of bothURAT1 and xanthine oxidase.

The improved biological activity profile of the compounds of theinvention and their potency make these compounds useful new drugs forreducing uric acid levels in blood, and for treating disorders of uricacid metabolism that are associated with, or caused by, elevated uricacid levels in blood or serum, including gout. Of particularsignificance is the advantage that the bifunctional compounds can beused effectively as monotherapy for reducing uric acid levels in blood,for treating or preventing disorders of uric acid metabolism, andspecifically for treating gout.

In a first aspect, compounds having a structure represented by Formula(I) are provided:

wherein

-   -   W, X, and Y are each independently O, S, NR² or N(R²)₂;    -   T is —CONR²—, —C(NR²)NH—, —C(NOR²)NH—, —C(N—NR²)NH—, —C(SR²)N—,        or —NHC(O)—;    -   A is phenyl, heteroaryl, C5-C10 branched or unbranched        cycloalkyl, C6-C10 bicycloalkyl or C5-C10 spirocycloalkyl;    -   each Z is independently present or absent and, if present, is        independently selected from one or more halogen atoms, —CN,        —CF₃, —OR², —C(O)R², SR², —S(O)_(g)R³ where g is 1 or 2,        —N(R²)₂, —NO₂, —CO₂R², —OCO₂R³, OC(O)R², —CON(R²)₂, —NR²C(O)R²,        —SO₂N(R²)₂, —NR²SO₂R³, —NR²SO₂N(R²)₂ or —NR²C(O)N(R²)₂,        —C(O)NHOR², alkyl, aryl, alkenyl and alkynyl;        wherein each R² is independently H, alkyl or aryl;        wherein each R³ is independently alkyl or aryl, optionally        substituted with one or more halogen atoms or OR²; and        wherein a, b, c, d, and e are each independently carbon or        nitrogen, or four of a, b, c, d, and e are each independently        carbon or nitrogen and one of a, b, c, d, and e is O, with the        proviso that at least one of a, b, c, d and e is nitrogen, and Z        is not connected directly to nitrogen or oxygen.

In one or more embodiments, the compound having a structure representedby Formula (I) is a compound wherein T is —CONR²—.

In one or more embodiments, the compound having a structure representedby Formula (I) is a compound wherein the 5-member heterocyclic ring is asubstituted or unsubstituted triazole, or a substituted or unsubstitutedoxadiazole.

Specific examples of compounds having a structure represented by Formula(I) include the following:

1. A compound wherein the 5-member heterocyclic ring is unsubstitutedtriazole. Representative examples of such compounds include:

-   -   The compound wherein A is phenyl, T is —C(N═NH)NH—; a is C; b is        CH; c and d are N; e is NH, and tautomers thereof, such as a        structure represented by Formula (I_(a)):

-   -   The compound wherein A is phenyl, T is —C(SCH₃)═N—; a is C; b is        CH; c and d are N; e is NH, and tautomers thereof, such as a        structure represented by Formula (I_(b)):

-   -   The compound wherein A is phenyl, T is —C(NOH)NH—; a is C; b is        CH; c and d are N; e is NH, and tautomers thereof, such as a        structure represented by Formula (I_(c)):

-   -   The compound wherein A is phenyl, T is —C(NNH₂)NH—; is C; b is        CH; c and d are N; e is NH, and tautomers thereof, such as a        structure represented by Formula (I_(d)):

-   -   The compound wherein A is phenyl, T is —C(O)NH—; both R² are        methyl (CH₃); a is C; b is CH; C; c and d are N; e is NH, and        tautomers thereof, such as a structure represented by Formula        (I_(c)):

-   -   The compound wherein A is phenyl, T is —C(NH)NH—; both R² are        methyl (CH₃); a is C; b is CH; c and d are N; e is NH, and        tautomers thereof, such as a structure represented by Formula        (I_(f)):

-   -   The compound wherein A is phenyl, T is —C(O)NH—; R² on the        3-position nitrogen is methyl (CH₃); R² on the 1-position        nitrogen is H; a is C; b is CH; c and d are N; e is NH, and        tautomers thereof, such as a structure represented by Formula        (I_(g)):

-   -   The compound wherein A is phenyl, T is —C(NH)NH—; R² on the        3-position nitrogen is methyl (CH₃); R² on the 1-position        nitrogen is H; a is C; b is CH; c and d are N; e is NH, and        tautomers thereof, such as a structure represented by Formula        (I_(h)):

-   -   The compound wherein A is unbranched cycloalkyl; T is —C(O)NH—;        a is C; b is CH; c is NH; d and e are N, and tautomers thereof,        such as a structure represented by Formula (I_(i)):

-   -   The compound wherein A is branched cycloalkyl; T is —C(O)NH—; a        is C; b is CH; c is NH; d and e are N, Z on the cyclohexyl ring        is OH, and tautomers thereof, such as a structure represented by        Formula (Ij):

-   -   The compound wherein A is phenyl, T is —C(O)NH—; Y is NH₂; a is        C; b is CH; c is NH; d and e are N, and tautomers thereof, such        as a structure represented by Formula (I_(k)):

and

-   -   The compound wherein A is phenyl, T is —NHC(O)—; a is C; b is        CH; c and d are N; e is NH, and tautomers thereof, such as a        structure represented by Formula (I_(l)):

2. A compound wherein the 5-member heterocyclic ring is substitutedtriazole. Representative examples of such compounds include:

-   -   The compound wherein A is phenyl, T is —C(O)NH—; X is S; W is o;        a is C; b is CH; c and d are N; e is NH; Z is phenyl on the        triazole ring, and tautomers thereof, such as a structure        represented by Formula (I_(m)):

3. A compound wherein the 5-member heterocyclic ring is substitutedoxadiazole. Representative examples of such compounds include:

-   -   The compound wherein A is phenyl, T is —C(O)NH—; a and c are C;        b and e are N; d is o; Z on the 5-member heterocyclic ring is        CF₃, and tautomers thereof, such as a structure represented by        Formula (I):

In a second aspect, compounds having a structure represented by Formula(II) are provided:

wherein

-   -   W, X, and Y are each independently O, S, NR² or N(R²)₂;    -   A is phenyl, heteroaryl, C5-C10 branched or unbranched        cycloalkyl, C6-C10 bicycloalkyl or C5-C10 spirocycloalkyl;    -   each Z is independently present or absent and, if present, is        independently selected from one or more halogen atoms, —CN,        —CF₃, —OR², —C(O)R², SR², —S(O)_(g)R³ where g is 1 or 2,        —N(R²)₂, —NO₂, —CO₂R², —OCO₂R³, OC(O)R², —CON(R²)₂, —NR²C(O)R²,        —SO₂N(R²)₂, —NR²SO₂R³, —NR²SO₂N(R²)₂ or —NR²C(O)N(R²)₂,        —C(O)NHOR², alkyl, aryl, alkenyl and alkynyl;        wherein each R¹ is C1-C8 branched or unbranched alkyl,        optionally substituted with Z;        wherein each R² is independently H, alkyl or aryl;        wherein each R³ is independently alkyl or aryl, optionally        substituted with one or more halogen atoms or OR²; and        wherein a, b, c, d, and e are each independently carbon or        nitrogen, or four of a, b, c, d, and e are each independently        carbon or nitrogen and one of a, b, c, d, and e is O, with the        proviso that at least one of a, b, c, d and e is nitrogen, and Z        is not connected directly to nitrogen or oxygen.

In one or more embodiments, the 5-member heterocyclic ring of thecompound having a structure represented by Formula (II) is a substitutedor unsubstituted triazole.

In one or more embodiments, the compound having a structure representedby Formula (II) is a compound wherein R¹ is —CH₃.

In one or more embodiments, the compound having a structure representedby Formula (II) is a compound wherein —XR¹ is —SCH₃ or —OCH₃.

Specific examples of compounds having a structure represented by Formula(II) include the following:

1. A compound wherein the 5-member heterocyclic ring is an unsubstitutedtriazole. Representative examples of such compounds include:

-   -   The compound wherein A is phenyl; X is S; R¹ and R² are methyl        (CH₃); a is C; b is CH; c and d are N; e is NH, and tautomers        thereof, such as a structure represented by Formula (II_(a)):

-   -   The compound wherein A is phenyl; X is O; R¹ is methyl (CH₃); R²        is H; a is C; b is CH; c and d are N; e is NH, and tautomers        thereof, such as a structure represented by Formula (II_(b)):

-   -   The compound wherein A is phenyl; X is S; R¹ is methyl (CH₃); R²        is H; a is C; b is CH; d and e are N; c is NH, and tautomers        thereof, such as a structure represented by Formula (II_(c)):

-   -   The compound wherein A is phenyl; X is S; R¹ is —C(CH₃)₂; R² is        H; a is C; b is CH; d and e are N; c is NH, and tautomers        thereof, such as a structure represented by Formula (II_(d)):

-   -   The compound wherein A is heteroaryl; X is S; R¹ is -methyl        (CH₃); R² is H; a is C; b is CH; d and e are N; c is NH, and        tautomers thereof, such as a structure represented by Formula        (II_(e)):

-   -   The compound wherein A is cycloalkyl; X is S; R¹ is -methyl        (CH₃); R² is H; a is C; b is CH; d and e are N; c is NH, and        tautomers thereof, such as a structure represented by Formula        (II_(f)):

2. A compound wherein the 5-member heterocyclic ring is a substitutedtriazole. Representative examples of such compounds include:

-   -   The compound wherein A is phenyl; X is S; R¹ is methyl (CH₃); R²        is H; a is C; b is C(CH3); d and e are N; c is NH, and tautomers        thereof, such as a structure represented by Formula (II_(g)):

-   -   The compound wherein A is phenyl; X is O; R¹ is methyl (CH₃); R²        is H; a is C; b is C(CH3); d and e are N; c is NH, and tautomers        thereof, such as a structure represented by Formula (II_(h)):

In a third aspect, compounds having a structure represented by Formula(III) are provided:

wherein

W, X, and Y are each independently O, S, NR² or N(R²)₂;

each R² is independently H, alkyl or aryl; and

f is divalent —CR²—, —C(O)—, —SR², —S(O)_(g)— where g is 1 or 2, —NR₂—,or —O(CR²)_(n)O— where n=2-3.

In one or more embodiments, the compound having a structure representedby Formula (III) is a compound wherein f is —S(O)_(g)—.

Specific examples of compounds having a structure represented by Formula(III) include the following:

1. A compound wherein X is O, R² is H, and f is —S(O)₂—, such as thecompound having a structure represented by Formula (III_(a)) andtautomers thereof:

In a fourth aspect, compounds having a structure represented by Formula(IV) are provided:

wherein

W, X, and Y are each independently O, S, NR² or N(R²)₂;

each R² is independently H, alkyl or aryl; and

f is divalent —CR²—, —C(O)—, —S(O)_(g)— where g is 1 or 2, —NR²—; or—C(—O(CR²)_(n)O—)— where n=2-3.

In one or more embodiments, the compound having a structure representedby Formula (IV) is a compound wherein f is —NH—, —C(—O(CR²)_(n)O—)—where n=2-3, or —C(O)—.

In one or more embodiments, the compound having a structure representedby Formula (IV) is a compound wherein f is —NH—, —C(—O(CR²)_(n)O—)— or—C(O)—, and X on the fused ring structure is O.

Specific examples of compounds having a structure represented by Formula(IV) include the following:

1. A compound wherein f is —NH (i.e., the fused ring structure isbenzodiazole). Representative examples of such compounds include:

-   -   The compound wherein X on the fused ring structure is O, such as        a structure represented by a Formula having a structure        represented by Formula (IV_(a)), and tautomers thereof:

2. A compound wherein f is —CO— or —C(—O(CR²)_(n)O—)— (i.e., the fusedring structure is benzopyrrolidine). Representative examples of suchcompounds include:

-   -   The compound wherein f is —C(O)— and X on the fused ring        structure is O, such as the compound having a structure        represented by Formula (IV_(b)), and tautomers thereof:

-   -   The compound wherein f is —C(—O(CR²)_(n)O—)— and X on the fused        ring structure is O, such as the compound having a structure        represented by Formula (IV_(c)), and tautomers thereof:

In a fifth aspect, compounds having a structure represented by Formula(V) are provided:

wherein

W, X, and Y are each independently O, S, NR² or N(R²)₂; and

each R² is independently H, alkyl or aryl.

In one or more embodiments, the compound having a structure representedby Formula (V) is a compound wherein each R² is H.

Specific examples of compounds having a structure represented by Formula(V) include the following:

1. A compound wherein each R² is H, X is O and W is O, such as thecompound having a structure represented by Formula (V_(a)), andtautomers thereof:

In a sixth aspect, compounds having a structure represented by Formula(VI) are provided:

wherein

-   -   W and X are each independently O, S, NR² or N(R²)₂;    -   A is phenyl, heteroaryl, C5-C10 branched or unbranched        cycloalkyl, C7-C10 bicycloalkyl or C5-C10 spirocycloalkyl;    -   each Z is independently present or absent and, if present, is        independently selected from one or more halogen atoms, —CN,        —CF₃, —OR², —C(O)R², SR², —S(O)_(g)R³ where g is 1 or 2,        —N(R²)₂, —NO₂, —CO₂R², —OCO₂R³, OC(O)R², —CON(R²)₂, —NR²C(O)R²,        —SO₂N(R²)₂, —NR²SO₂R³, —NR²SO₂N(R²)₂ or —NR²C(O)N(R²)₂,        —C(O)NHOR², alkyl, aryl, alkenyl and alkynyl;    -   U is —O—, —S— —NR²— or —S(O)_(g)— where g is 1 or 2;        wherein each R² is independently H, alkyl or aryl;        wherein each R³ is independently alkyl or aryl, optionally        substituted with one or more halogen atoms or OR²; and        wherein a, b, c, d, and e are each independently carbon or        nitrogen, or four of a, b, c, d, and e are each independently        carbon or nitrogen and one of a, b, c, d, and e is O, with the        proviso that at least one of a, b, c, d and e is nitrogen, and Z        is not connected directly to nitrogen or oxygen.

In one or more embodiments, the compound having a structure representedby Formula (VI) is a compound wherein U is —O— or —NH—.

In one or more embodiments, the compound having a structure representedby Formula (VI) is a compound wherein the 5-member heterocyclic ring istriazole.

Specific examples of compounds having a structure represented by Formula(VI) include the following:

1. A compound wherein the 5-member heterocyclic ring is triazole and Uis O. Representative examples of such compounds include:

-   -   The compound wherein X and W are O, such as the compound having        a structure represented by Formula (VI_(a)), and tautomers        thereof.

2. A compound wherein the 5-member heterocyclic ring is triazole and Uis —NH—. Representative examples of such compounds include:

-   -   The compound wherein R² on both nitrogens of the barbiturate        ring is —H, such as a structure represented by Formula (VI_(b)),        and tautomers thereof:

and

-   -   The compound wherein R² on both nitrogens of the barbiturate        ring is —CH₃, such as a structure represented by Formula        (VI_(c)), and tautomers thereof:

In a seventh aspect, compounds having a structure represented by Formula(VII) are provided:

wherein

-   -   X and Y are each independently O, S, NR² or N(R²)₂;    -   Z is present or absent and, if present, is selected from one or        more halogen atoms, —CN, —CF₃, —OR², —C(O)R², SR², —S(O)_(g)R³        where g is 1 or 2, —N(R²)₂, —NO₂, —CO₂R², —OCO₂R³, OC(O)R²,        —CON(R²)₂, —NR²C(O)R², —SO₂N(R²)₂, —NR²SO₂R³, —NR²SO₂N(R²)₂ or        —NR²C(O)N(R²)₂, —C(O)NHOR², alkyl, aryl, alkenyl and alkynyl,        wherein each R² is independently H, alkyl or aryl; and        wherein a, b, c, d, and e are each independently carbon or        nitrogen, or four of a, b, c, d, and e are each independently        carbon or nitrogen and one of a, b, c, d, and e is O, with the        proviso that at least one of a, b, c, d and e is nitrogen, and Z        is not connected directly to nitrogen or oxygen.

In one or more specific embodiments, the compound having a structurerepresented by Formula (VII) is a compound wherein the unfused 5-memberheterocyclic ring is triazole.

Specific examples of compounds having a structure represented by Formula(VII) include the following:

1. A compound wherein the unfused 5-member heterocyclic ring is triazoleand each R² is H. Representative examples of such compounds include:

-   -   The compound wherein a is C; b is CH; c and d are N and e is NH,        such as the compound having a structure represented by Formula        (VII_(a)), and tautomers thereof:

In an eighth aspect, compounds having a structure represented by Formula(VIII) are provided:

wherein

-   -   W, X, and Y are each independently O, S, NR² or N(R²)₂;    -   T is —CONR²—, —C(NR²)NH—, —C(NOR²)NH—, —C(N—NR²)NH—, —C(SR²)N—,        or —NHC(O)—;    -   A is phenyl, heteroaryl, C5-C10 branched or unbranched        cycloalkyl, C6-C10 bicycloalkyl or C5-C10 spirocycloalkyl;    -   each Z is independently present or absent and, if present, is        independently selected from one or more halogen atoms, —CN,        —CF₃, —OR², —C(O)R², SR², —S(O)_(f)R³ where f is 1 or 2,        —N(R²)₂, —NO₂, —CO₂R², —OCO₂R³, OC(O)R², —CON(R²)₂, —NR²C(O)R²,        —SO₂N(R²)₂, —NR²SO₂R³, —NR²SO₂N(R²)₂ or —NR²C(O)N(R²)₂,        —C(O)NHOR², alkyl, aryl, alkenyl and alkynyl;        wherein each R² is independently H, alkyl or aryl;        wherein each R³ is independently alkyl or aryl, optionally        substituted with one or more halogen atoms or OR².

In one or more specific embodiments, the compound having a structurerepresented by Formula (VIII) is a compound wherein Z is —C(O)NHOR².

Specific examples of compounds having a structure represented by Formula(VIII) include the following:

1. A compound wherein Z is —C(O)NHOR². Representative examples of suchcompounds include:

-   -   The compound wherein A is phenyl, such as the compound having a        structure represented by Formula (VIIIa), and tautomers thereof:

and

-   -   The compound wherein A is heteroaryl (for example pyridine),        such as the compound having a structure represented by Formula        (VIII_(b)), and tautomers thereof:

As disclosed herein, reference to compounds having a structurerepresented by any of Formulae (I)-(VIII), or a combination thereof, isintended to include all compounds falling within the generic structure,as well as the specific embodiments described and their tautomers.

The compounds disclosed herein can be synthesized various generalprocedures, as depicted in FIGS. 1-11. In general the various syntheticroutes center on the coupling of a substituted phenyl, heterocyclic,cycloalkyl or spirocyclic A ring with an appropriately substitutedbarbiturate ring. Several different coupling agents can be employed inthis process and the general procedures are described below in “GeneralProcedure 1” or “General Procedure 2”. Many of the compounds can be madeas illustrated in FIG. 1, if the appropriately substituted nitro oramino ring A is known in the art. Variations to this sequence of stepsare shown in FIGS. 2-11. The syntheses of certain bridged systems areillustrated in FIG. 2. This process requires the introduction of aketene thio acetyl, wherein the sulfur substituents can then bedisplaced with various amines, like ring A containing an amino group,followed by a substituted hydrazine or hydroxylamine. Subsequent ringclosure is accomplished by known procedures. FIG. 3 depicts anotherpossible route to make certain of the compounds. This process centers onconstruction of the barbiturate ring at the end of the synthesis viatreatment of a substituted malonate with a substituted urea or thiourea.FIG. 4 relates to synthesis of compounds that contain a hydroxamic acidin place of the heterocyclic ring. The process involves addition of acompound containing an ester on the A ring. This ester is then readilyconverted to a hydroxamic acid after coupling to the barbiturate ringvia known procedures. FIG. 5 depicts the synthesis of compounds thatcontain a fused heterocycle on the A ring. In general the fusedheterocycle is prepared with an amino group on the A ring and then it iscoupled to the barbiturate ring as described above. FIGS. 6-8 depictmethods of synthesis that result in compounds containing a substituenton X of the barbiturate ring. FIG. 6 is the most straightforward way ofmaking such compounds, as it generally follows the sequence depicted inFIG. 1. However, this method involves a subsequent last step, whichinvolves alkylation of the X group. This is only possible if R² is analkyl group, and separation of the various possible isomers obtained maybe necessary. The synthesis outlined in FIG. 7 is more direct in that itensures that the R² group is attached to the barbiturate ring. Thisprocess involves the introduction of the R² group early in thesynthesis, first by condensation of malonate to an appropriatelysubstituted urea, followed by alkylation, which again is conducted withan alkyl halide (i.e., R² is alkyl). In certain cases, a protectinggroup may be necessary depending on the nature of the substituents. Inanother case the R² group on the X can be introduced in the earliestpart of the synthesis. It can be intact on the substituted urea orthiourea. This would allow for compounds that contain an aryl orheterocyclic aryl group on X. This is depicted in FIG. 8 and involvesthe condensation of the R² substituted urea or thiourea with malonate.The subsequent barbiturate ring is then coupled to the appropriate aminoA ring compound to produce the desired product. FIG. 9 depicts anothervariation of synthesis of the bridged compounds described in FIG. 2. Itinvolves formation of a hydrazine A ring first, followed by condensationwith methyl 4,6-dichloropyrimidine-5-carboxylate. Workup not onlyprovides the cyclized bridged ring system, but also oxidizes theposition adjacent to the two nitrogens on the pyrimidine ring. FIG. 10depicts methods for forming the triazole heterocyclic ring when it isnot known in the art. Addition of azide to an amino containing A ringcan be accomplished via a variety of methods which all involve theaddition of azide to the acetylene. Protecting groups as illustrated inthe figure may be necessary. Sometimes the acetylene containing A ringmay not be known in the art, so it needs to be synthesized. This can beaccomplished via a variety of methods as illustrated in FIG. 11.Treatment of the corresponding aldehyde of the ring A compound with1-diazo-1-((dimethylperoxy)(oxo)-λ⁴-phosphanyl)propan-2-one, which isknown in the art, or Sonogashira reaction on the halide of a ring Acompound would produce the corresponding acetylenic A ring containingcompound. Subsequent addition of azide to the acetylene would producethe triazole. This can then be coupled by the methods described above toproduce the desired targeted compound.

In one aspect, the invention provides methods for reducing uric acidlevels in the blood or serum of a subject comprising administering acompound having a structure represented by any of Formulae (I)-(VIII),or a combination thereof, to the subject in an amount effective toreduce blood or serum uric acid levels. It is to be understood that allsuch methods for reducing uric acid levels correspond to a compoundhaving a structure represented by any of Formulae (I)-(VIII), or acombination thereof, for use in medicine as well as a compound having astructure represented by any of Formulae (I)-(VII), or a combinationthereof, for use in the treatment of elevated uric acid levels.Typically, the compound having a structure represented by, or acombination thereof, will be administered when the level of uric acid inthe blood of the subject is elevated, i.e., in the upper range of normalor above normal levels. One skilled in the art would further recognizethat continued administration after normal uric acid levels are achievedis also contemplated in order to maintain uric acid levels within thenormal range and to reduce the overall body burden of uric acid that mayhave occurred due to previously sustained hyperuricemia. Accordingly,methods for preventing elevation of uric acid levels in blood or serumare also an aspect of the invention. It is to be understood that allsuch methods for preventing elevation of uric acid levels correspond toa compound having a structure represented by any of Formulae (I)-(VIII),or a combination thereof, for therapeutic use as well as a compoundhaving a structure represented by any of Formulae (I)-(VIII), or acombination thereof, for prevention of elevated uric acid levels.

Normal uric acid levels in blood are generally in the range of 4.3 mg/dLto 8.0 mg/dL. In certain embodiments, a compound having a structurerepresented by any of Formulae (I)-(VIII), or a combination thereof, isadministered to a subject with a blood uric acid level of at least about6 mg/dL. Administration may continue until a blood uric acid level ofabout 6.0 mg/dL or less is reached; however, it is generally consideredto be beneficial to maintain uric acid levels below this target inpatients with disorders of uric acid metabolism.

In certain embodiments, the invention provides methods of treating adisorder of uric acid metabolism caused by, or associated with, elevateduric acid levels in blood or serum (hyperuricemia). The method oftreating such disorders comprises administering a compound having astructure represented by any of Formulae (I)-(VIII), or a combinationthereof, to a subject in need thereof in an amount effective to reduceserum uric acid levels, thereby treating the disorder of uric acidmetabolism in the subject. These disorders are associated with, orcaused by, elevated uric acid levels in blood or serum which are in theupper range of normal or above normal, and include gout, hyperuricemia,kidney disease, arthritis, kidney stones, kidney failure, urolithiasis,plumbism, hyperparathyroidism, psoriasis, inborn genetic errors ofmetabolism (including but not limited to Lesch-Nyhan syndrome),sarcoidosis, cardiovascular disease (including but not limited toatherosclerosis), and transplantation of blood, bone marrow or solidorgans. These drugs are particularly useful for treating gout and kidneydisease (including acute uric acid nephropathy, chronic uratenephropathy, and uric acid nephrolithiasis). In addition, treatment ofsome cancers with chemotherapy leads to the release of large amounts ofuric acid into the blood, which can damage the kidneys.Chemotherapy-induced hyperuricemia, particularly the disorder known as“tumor lysis syndrome,” may also be treated, prevented or amelioratedaccording to the methods of the invention. Administration of a compoundhaving a structure represented by any of Formulae (I)-(VIII), or acombination thereof, to a subject with hyperuricemia, such as a subjectsuffering from gout, kidney disease, or a risk of inducing elevated uricacid levels due to chemotherapy, treats, prevents or ameliorates thesedisorders by reducing uric acid levels in blood, or preventing orcontrolling their level of increase. In specific embodiments, thedisorder of uric acid metabolism treated by administration of a compoundhaving a structure represented by any of Formulae (I)-(VIII), acombination thereof, is gout. It is to be understood that all suchmethods for treating disorders of uric acid metabolism caused by, orassociated with, elevated uric acid levels in blood or serum(hyperuricemia) correspond to a compound having a structure representedby any of Formulae (I)-(VIII), or a combination thereof, for therapeuticuse as well as a compound having a structure represented by any ofFormulae (I)-(VIII), or a combination thereof, for treatment ofdisorders of uric acid metabolism caused by, or associated with,elevated uric acid levels in blood or serum.

The dose of a compound having a structure represented by any of Formulae(I)-(VIII), or a combination thereof, administered to the subject may beany dose sufficient to achieve a desired reduction in uric acid levelsin blood or serum over the time-course of administration. In certainembodiments, a daily dose of about 20 to about 1,500 mg/m²/day isadministered. In other embodiments, a daily dose of about 20 to about500 mg/m²/day, about 20 to about 250 mg/m²/day, about 20 to about 150mg/m²/day or about 20 to about 100 mg/m²/day is administered. In otherembodiments, a daily dose of about 50 to about 1,500 mg/m²/day isadministered. In other embodiments, a daily dose of about 50 to about500 mg/m²/day, about 50 to about 150 mg/m²/day, about 50 to about 100mg/m²/day, or about 20 to about 100 mg/m²/day is administered.

In certain embodiments of any of the foregoing methods, a compoundhaving a structure represented by any of Formulae (I)-(VIII), or acombination thereof, is administered to the subject parenterally,intraperitoneally, intravenously, intranasally, intrarectally, ororally. Particularly useful routes of administration include injection,infusion, or oral administration. The amount of the drug administeredper dose is an amount sufficient to achieve a reduction in uric acidlevels in blood or serum, to prevent elevation of uric acid levels inblood or serum, or to treat or prevent a disorder of uric acidmetabolism over the course of therapy. One skilled in the art willrecognize that individualization of dosage based on a patient's bodycomposition or his/her hypouricemic response to treatment may bemedically necessary or desirable.

The drug(s) may be administered to the subject either intermittently orcontinuously over a period of time in order to achieve the desiredreduction in uric acid levels in blood or serum, or to treat a disorderof uric acid metabolism. For example, doses may be administeredintermittently several times per day, daily, once, twice or three timesper week, or at monthly intervals. In a specific example, a compoundhaving a structure represented by any of Formulae (I)-(VIII), or acombination thereof, may be administered to the subject by continuousintravenous infusion over 24 hours for about five days. Alternatively, acompound having a structure represented by any of Formulae (I)-(VIII),or a combination thereof, may be administered to the subject byintravenous infusion over about 1 hour to about 5 hours for about fiveconsecutive days. In a specific example, a compound having a structurerepresented by any of Formulae (I)-(VIII), or a combination thereof, maybe administered to the subject by intramuscular injection or byintravenous infusion over about 10 minutes for about five consecutivedays. In further specific embodiments, a compound having a structurerepresented by any of Formulae (I)-(VIII), or a combination thereof, maybe administered to the subject by daily bolus injections for about fivedays. The period of time of administration in any of the foregoingprotocols may be modified to achieve the desired reduction in uric acidlevels, including about 2 days, about 3 days, about 4 days, about oneweek or about two weeks of administration, or for longer periods inrepeated treatment cycles, and these treatments may be repeated atintervals of every two to every 10 weeks.

In addition to continuous intravenous infusion or bolus intravenous orsubcutaneous injection, a compound having a structure represented by anyof Formulae (I)-(VIII), or a combination thereof, may be administered tothe subject orally. In this embodiment, an oral dose in amounts asdescribed above may be administered in one, two, three or fouradministrations per day for 1, 2, 3, 4, or 5 days to achieve the desiredreduction in uric acid levels. In further embodiments, the oral dose asdescribed above may be administered once per day, or in one, two, threeor four administrations per day for one week or two weeks, to achievethe desired reduction in uric acid levels.

It will be appreciated that a subject in need of reduced levels of uricacid in blood or serum, or in need of treatment of a disorder of uricacid metabolism, will be treated more aggressively initially to achievethe desired reduction in uric acid levels. Following initial therapy andreduction of uric acid levels to normal or sub-normal levels, thesubject may be further treated over a period of time, or over alifetime, to maintain normal or sub-normal levels of uric acid in bloodor serum and prevent elevation of uric acid levels subsequent to theinitial treatment. The maintenance or preventive protocol may comprisereduced dosages and/or less frequent administration of a compound havinga structure represented by any of Formulae (I)-(VIII), or a combinationthereof, as necessary or desired to maintain normal or sub-normal uricacid levels in blood or serum. For example, in a maintenance protocolthe drug(s) may be administered daily, weekly, monthly, orintermittently as uric acid levels rise between treatment periods. Suchmaintenance protocols will serve to maintain normal or sub-normal uricacid levels for a prolonged period of time and reduce the subject'slifetime risk of developing a disorder of uric acid metabolism causedby, or associated with, prolonged hyperuricemia. The initial reductionof uric acid levels from above normal or high normal to normal orsub-normal, and maintenance of normal or sub-normal uric acid levels areboth features included in treatment of a disorder of uric acidmetabolism. It is anticipated that in certain embodiments, a typicalpatient will require daily treatment of varying duration, and that suchdaily treatment may be provided intermittently for life or for extendedperiods.

In certain embodiments of any of the foregoing methods, blood or serumuric acid levels of the subject are reduced by at least 25% compared touric acid levels prior to administration of a compound having astructure represented by any of Formulae (I)-(VIII), or a combinationthereof. In certain further embodiments, blood or serum uric acid levelsof the subject are reduced by 50% or more compared to levels prior toadministration. In a specific embodiment, uric acid levels are reducedby about 75% even at daily doses of 500 mg/m²/day or less.

In a second aspect of the invention methods are provided for treating adisorder of uric acid metabolism associated with, or caused by, elevateduric acid in blood or serum comprising administering to a subject inneed thereof a compound having a structure represented by any ofFormulae (I)-(VIII), or a combination thereof, in an amount effective toreduce blood or serum uric acid levels, thereby treating the disorder ofuric acid metabolism. Specific embodiments of the methods for treating adisorder of uric acid metabolism relating to dosing, routes ofadministration, initial therapy and maintenance therapy are as describedabove for reducing uric acid levels in blood or serum. The initialreduction in uric acid levels is typically rapid, and often occurswithin 1-3 days. Upon reduction in uric acid levels to normal orsub-normal levels, continued maintenance or preventive therapy resultsin a detectable improvement in at least one symptom of elevated uricacid, for example reduced inflammation, reduced pain, slowing ofdevelopment of deformities, reduced development of kidney stones,prevention of tumor lysis syndrome, stabilization in cognition or othermanifestations of inborn metabolic disorders, or improvement in (orreduction of actual or risk for) cardiovascular disease. One skilled inthe art will recognize that prevention of recurrent symptoms of diseasedue to recurrence of elevated serum uric acid levels, therebynecessitating extended treatment, would be highly desirable to maximizepatient benefit.

In embodiments corresponding to the foregoing methods, the inventionrelates to use of a compound disclosed herein, or a combination thereof,for reducing uric acid levels in blood or serum of a subject in needthereof, preventing elevation of uric acid levels in blood or serum of asubject, or treating a disorder of uric acid metabolism caused by, orassociated with, hyperuricemia. Each of the methods of treatment orprevention disclosed, including routes of administration, dosage andcompounds administered, are also applicable to such uses of thecompounds.

A further aspect of the invention provides a pharmaceutical compositioncomprising a compound having a structure represented by any of Formulae(I)-(VIII), or a combination thereof, and a pharmaceutically acceptablecarrier. In certain embodiments of the pharmaceutical compositions, thecomposition is formulated as a solution or tablet. Solutions ordispersions of the drug(s) can be prepared in water or saline. Incertain embodiments of the pharmaceutical compositions, thepharmaceutically acceptable carrier is one or more component selectedfrom the group consisting of one or more of a solvent, a dispersingagent, a coating (e.g., lecithin), a surfactant (e.g.,hydroxypropylcellulose), a preservative (e.g., paraben, phenol,thimerosal, sorbic acid, chlorobutanol), an emulsion, an alcohol (e.g.,ethanol), a polyol (e.g., glycerol, propylene glycol), and an isotonicagent (e.g., sugars, sodium chloride).

In certain embodiments of the foregoing pharmaceutical compositions, thecomposition is formulated for controlled release of the compound havinga structure represented by any of Formulae (I)-(VIII), or a combinationthereof. In certain embodiments of the foregoing methods, a compoundhaving a structure represented by any of Formulae (I)-(VIII), or acombination thereof, is administered in a form for controlled release.The controlled release compositions may include pharmaceuticallyacceptable carriers or excipients which cause release of the activeingredient more slowly or which extend the duration of its action withinthe body. Examples of controlled release compositions includepharmaceutically acceptable carriers or excipients which delayabsorption of the active ingredient (e.g., aluminum monostearate,gelatin, natural or synthetic hydrophilic gums). Alternatively,controlled release of the pharmaceutical composition may employ a devicesuch as a pump, implant or transdermal patch.

In certain embodiments of the foregoing pharmaceutical compositions, thecomposition is formulated for improved oral bioavailability or extendedrelease in the body. For example, microemulsions, particle sizereduction and complexation technologies may be used to improvedissolution rates or equilibrium solubilities of the compounds. Othersuitable chemical and physical means for improving oral bioavailabilityor extended release will also be known to those skilled in the art.

EXAMPLES

General Procedure for carbonyldiimidazole (CDI) Coupling (GeneralProcedure 1): To a stirring solution of amine (1 eq) in anhydrous DMSO(1.0 M) was added 1,1′carbonyldiimidazole (1.5 eq) at rt, under inertatmosphere. The resulting solution was stirred for 20 min at rt. In aseparate flask containing barbituric acid (1 eq) was added anhydrous1,4-dioxane (0.30 M), then heated to 55° C. Et₃N (1.6 eq) was added andstirred for 15 min at 55° C. The isocyanate generated from the amine inDMSO was added to the stirring suspension, then heated to 80° C. untilcomplete consumption of the starting materials were observed via LCMS(1-20 h). The reaction mixture was cooled to rt, then acidified with 6MHCl (aq), the precipitate formed was isolated, then triturated withwater, MeOH, followed by acetonitrile.

General Procedure for Triphosgene coupling (General Procedure 2):2-(Methylthio)pyrimidine-4,6-diol (2 eq) was added to a stirringsolution of sodium tert-butoxide (2.0 eq) dissolved in DMSO (0.2 M) atrt for 5 min. In a separate flask, amine was dissolved in 1,4-dioxane(0.8 M), to this solution was added triphosgene (0.33 eq) inone-portion. The suspension was stirred vigorously for 2 min at rt, theniPr₂NEt (2 eq) was added. The suspension was stirred vigorously at rtfor 2 min. Freshly prepared solution of sodium6-hydroxy-2-(methylthio)pyrimidin-4-olate in DMSO was added to thesuspension in one-portion. The reaction was stirred at 90° C. for 30min, until complete consumption of starting material observed via LCMS.The reaction mixture was loaded directly on C18 column and purified viareverse-phase chromatography.

Example I: Preparation ofN-(4-(1H-1,2,3-triazol-4-yl)phenyl)-6-hydroxy-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboximidamide(11, Formula (I_(a)), with Reference to FIG. 12)

5-((4-(1H-1,2,3-triazol-4-yl)phenylamino)(methylthio)methylene)pyrimidine-2,4,6(1H,3H,5H)-trione(PA6) (48 mg, 0.140 mmol) was added a solution of 7.0 M NH₃ in methanol(1.0 mL) at rt. The reaction was sealed and heated to 60° C., untilcomplete consumption of the starting material was observed via LCMS (20h). The reaction mixture was allowed to cool to rt, the precipitateformed was filtered and washed with acetonitrile, then the solid waslyophilized to yield the product (11) as an off-white solid (39 mg,99.5% purity, 89% yield).

¹H NMR (500 MHz, DMSO-d6+DCl in D₂O) δ 8.49 (s, 1H), 7.92-7.87 (m, 2H),7.32-7.28 (m, 2H).

LCMS: m/z [M+1]⁺=314.09; R_(T)=0.93 min; purity=99.5%.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

Example 2: Preparation of4-(1H-1,2,3-triazol-4-yl)-N-(2,4,6-trioxohexahydropyrimidin-5-yl)benzamide(12, Formula (I₁), with Reference to FIG. 13)

Step One. Diethyl 2-(4-(1H-1,2,3-triazol-4-yl)benzamido)malonate (PA7).4-(1H-1,2,3-triazol-4-yl)benzoic acid (76 mg, 0.41 mmol, prepared asdescribed, in: Jin, T.; Kamijo, S.; Yamamoto, Y. Eur. J. Org. Chem.2004, 3789-3791), 2-amino-diethyl malonate HCl salt (106 mg, 0.48 mmol)and HATU (183 mg, 0.483 mmol) was sequentially added into DMF (2 mL),iPr₂NEt (156 mg, 1.2 mmol) was added to the mixture and the reaction wasstirred at rt for 1 h. The reaction mixture poured into water (20 mL)and the product was extracted with EtOAc (20 mL). The organic layer waswashed with water (20 mL) and brine (10 mL), dried over MgSO₄, thenconcentrated in vacuo. The product was purified via ISCO (0% to 10% MeOHin CH₂Cl₂) to yield the product (120 mg, 86% yield).

Step Two.4-(1H-1,2,3-triazol-4-yl)-N-(2,4,6-trioxohexahydropyrimidin-5-yl)benzamide(12). Diethyl 2-(4-(1H-1,2,3-triazol-4-yl)benzamido)malonate (PA7) (356mg, 1.03 mmol) and urea (50 mg, 0.82 mmol) dissolved in MeOH (10 mL). Tothe solution was added NaOMe (0.55 mL, 30% in MeOH). The mixture wasrefluxed overnight. To the reaction was added formic acid until neutral.The solvent was removed in vacuo and the crude was dissolved in DMSO (2mL). The crude product was purified via ISCO (C18, 5 to 100%acetonitrile in water, with an ammonium formate buffer 10 mM, over 15 CVgradient) to yield the product (12) as an off-white solid (62.0 mg,97.7% purity, 19.2% yield), after lyophilization.

¹H NMR (500 MHz, CDCl₃+DCl in D₂O) δ 8.52 (s, 1H), 8.06-7.99 (m, 4H).

LCMS: m/z [M-H]⁻=315.10; R_(T)=0.74 min; purity=97.7%.

HPLC conditions: Column: XTerra RP18, 3.5 μm, 3.0×50 mm; Gradient: 5% Bfor 0.3 min, 5% to 100% B in 4.5 minutes; 100% B for 2.2 minute; 1mL/min; 7 min run. Eluent A: Milli-Q H₂O+0.1% Formic Acid; Eluent B:Acetonitrile+0.1% Formic Acid.

Example 3: Preparation of5-(((4-(1H-1,2,3-triazol-5-yl)phenyl)amino)(methylthio)methylene)pyrimidine-2,4,6(1H,3H,5H)-trione(Formula (I_(b)), with Reference to FIG. 14)

Step One. 5-(Bis(methylthio)methylene)pyrimidine-2,4,6(1H,3H,5H)-trione(PA5). A mixture of barbituric acid (2.5 g, 21.4 mmol,), triethylamine(4.3 g, 42.7 mmol), and carbon disulfide (1.6 g, 21.4 mmol) was allowedto stir at rt until complete consumption of the starting material wasobserved via LCMS (0.5 h). The resulting reaction mixture was cooled inan ice-bath, iodomethane (6.1 g, 42.7 mmol) was added in one-portion,the reaction was then allowed to warm up to rt over 3 h. The reactionmixture was poured into water (150 mL) with rapid stirring, then allowedto stand for 1 h. The precipitate formed was filtered, washed with waterand acetone. The precipitate was collected and dried under high vacuumto yield the product as a yellow solid (750 mg, 15.1% yield).

Step Two.5-(((4-(1H-1,2,3-Triazol-4-yl)phenyl)amino)(methylthio)methylene)pyrimidine-2,4,6(1H,3H,5H)-trione(13). A mixture of PA5 (76.9 mg, 0.331 mmol) and XX (53 mg, 0.331 mmol)in anhydrous 1,4-dioxane (5 mL) was sealed in a microwave vessel andheated to 100° C. for 30 min, complete consumption of the startingmaterial was observed via LCMS. Evaporate the solvent in vacuo anddissolved the crude in DMSO. ISCO purification (C18, 10 mM AmF inwater/MeCN) was performed to yield the product (13) as a solid (49 mg,95.6% purity, 43% yield), after lyophilization.

¹H NMR (500 MHz, DMSO) δ 8.41 (s, 1H), 7.96 (d, J=8.6 Hz, 2H), 7.46 (dd,J=8.5, 2.6 Hz, 2H), 2.09 (s, 3H).

LCMS: m/z [M+1]⁺=343.08; R_(T)=3.29 min; purity=95.6%.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

Example 4: Preparation of(E)-N-(4-(1H-1,2,3-triazol-5-yl)phenyl)-N′-hydroxy-2,4,6-trioxohexahydro-pyrimidine-5-carboximidamide(14, Formula (I_(c)), with Reference to FIG. 15)

5-(((4-(1H-1,2,3-triazol-5-yl)phenyl)amino)(methylthio)methylene)pyrimidine-2,4,6(1H,3H,5H)-trione13 (180 mg, 0.60 mmol) and hydroxylamine (400 uL, 6.0 mmol, 50% inwater) were added into 1,4-dioxane (10 mL). The reaction was heated to100° C. for 30 min. The solvent was removed in vacuo and the crudemixture was dissolved in DMSO (2 mL). The crude product was purified viaISCO (C18, 5 to 100% acetonitrile in water, with an ammonium formatebuffer 10 mM, over 15 CV) to yield the product (14) as a yellow solid(41 mg, 94.0% purity, 24% yield), after lyophilization.

LCMS: m/z [M−1]⁻=328.07; R_(T)=2.90 min.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

Example 5: Preparation of3-((4-(1H-1,2,3-triazol-5-yl)phenyl)amino)isoxazolo[5,4-d]pyrimidine-4,6(5H,7H)-dione(16, Formula (VI_(a)), with Reference to FIG. 16)

Compound 14 (100 mg, 0.30 mmol) and DMFDMA (108 mg, 0.92 mmol) was addedinto anhydrous 1,4-dioxane (6 mL). The reaction mixture was heated up at50° C. After 30 min, another portion of DMFDMA (108 mg, 0.92 mmol) wasadded to the reaction mixture. Reaction was stirred at the sametemperature for an additional 30 min. The solvent was removed and thecrude product was dissolved in DMSO (2 mL) and purified via Prep HPLCseparation (0 to 100% acetonitrile in water with 10 mM ammonium formatebuffer, over 10 min) to yield the product (16) as a white solid (24 mg,99.5% purity, 28% yield), after lyophilization.

¹H NMR (500 MHz, d⁶-DMSO+DCl) δ 8.29 (s, 1H), 7.84 (d, J=8.9 Hz, 2H),7.71 (d, J=8.9 Hz, 2H).

LCMS: m/z [M−1]⁻=310.11; R_(T)=3.23 min; purity=99.5%

HPLC conditions: Waters XTerra RP18 3.5 μm, 3.0×50 mm; hold 5% B for 1.0minute, 5% to 95% B in 5.0 minutes, then hold 5% B for 1.0 minute, runtime=7.0 min; Eluents: A=0.1% HCO₂H in water; B=0.1% HCO₂H in MeCN.

Example 6: Preparation of(E)-N-(4-(1H-1,2,3-triazol-5-yl)phenyl)-2,4,6-trioxohexahydropyrimidine-5-carbohydrazonamide(17, Formula (I_(d)), with Reference to FIG. 17)

Compound 13 (100 mg, 0.33 mmol) and hydrazine hydrate solution (165 μL,3.3 mmol, 55% in H₂O) was added into 1,4-dioxane (10 mL, dry)sequentially. The reaction was heated to 105° C. for 30 min. Theprecipitate was filtered to yield the product (17) as a beige solid (180mg, 96.8% purity, 83% yield).

¹H NMR (500 MHz, d⁶-DMSO+DCl In D₂O) δ 10.68 (s, 1H), 8.37 (s, 1H), 7.84(d, J=8.6 Hz, 2H), 7.27 (d, J=8.6 Hz, 2H),

LCMS: m/z [M−1]⁻=327.22; R_(T)=2.50 min; purity=96.8%.

HPLC conditions: Waters XTerra RP18 3.5 μm, 3.0×50 mm; hold 5% B for 1.0minute, 5% to 95% B in 5.0 minutes, then hold 5% B for 1.0 minute, runtime=7.0 min; Eluents: A=0.1% HCO₂H in water; B=0.1% HCO₂H in MeCN.

Example 7: Preparation ofN-(4-(1H-1,2,3-triazol-4-yl)phenyl)-6-hydroxy-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide(19, Formula (I_(c)), with Reference to FIG. 18)

Compound 19 was synthesized following general procedure 1. To a stirringsolution of XX (27 mg, 0.169 mmol) in anhydrous DMSO (170 μL) was added1,1′carbonyldiimidazole (41 mg, 0.254 mmol) was added at rt, under inertatmosphere. The resulting solution was stirred for 20 min at rt. In aseparate flask containing 1,3-dimethylbarbituric acid (23 mg, 0.169mmol) was added anhydrous 1,4-dioxane (506 μL), then heated to 50° C.Et₃N (37 μL, 0.27 mmol) was added and stirred for 15 min at 50° C. Theisocyanate generated from XX in DMSO was added to the stirringsuspension, then heated to 80° C. until complete consumption of thestarting materials were observed via LCMS (1 h). The reaction mixturewas cooled to rt, then acidified with 6M HCl (aq), the precipitateformed was isolated, then triturated with MeOH, followed byacetonitrile, to yield the product (19) as an off-white solid (5.6 mg,97.8% purity, 4% yield).

¹H NMR (500 MHz, DMSO-d6) δ 8.29 (s, 1H), 7.88 (s, 2H), 7.66 (d, J=8.8Hz, 2H), 3/24 (s. 6H).

LCMS: m/z [M+1]⁺=343.57; R_(T)=2.51 min; purity=97.8%.

HPLC conditions: Column: XTerra RP18, 3.5 μm, 3.0×50 mm; Gradient: 5% to100% B in 2.5 minutes; 100% B for 1 minute; 1 mL/min; 4 min run. EluentA: Milli-Q H₂O+0.1% Formic Acid; Eluent B: Acetonitrile+0.1% FormicAcid.

Example 8: Preparation of6-hydroxy-N-(4-hydroxy-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide(20, Formula (V_(a)), with Reference to FIG. 19)

Step One. 4-Chloro-1H-pyrazolo[3,4-d]pyrimidin-6-amine (PA17). Hydrazinehydrate solution (295 μL, 5.21 mmol, 55% in H₂O) was added dropwise to astirring suspension of 2-amino-4,6-dichloropyrimidine-5-carbaldehyde(1.00 g, 5.21 mmol) and triethylamine (835 mL, 5.99 mmol) in THF (21 mL)and H₂O (2.1 mL) at rt. The reaction was allowed to stir for 4 h, thenconcentrated. The residue was added H₂O, the precipitate was washed withwater, then collected and dried under high vacuum to yield the productas a yellow solid (765 mg, 87% yield).

¹H NMR (500 MHz, DMSO-d6) δ 7.94 (s, 1H), 7.12 (s, 2H), 4.09 (s, 1H).

LCMS: m/z [M+1]⁺=169.98; R_(T)=0.79 min.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

Step Two. 6-amino-1H-pyrazolo[3,4-d]pyrimidin-4-ol (PA18).4-Chloro-1H-pyrazolo[3,4-d]pyrimidin-6-amine (PA17) (365 mg, 2.16 mmol)was added 2M NaOH (5.7 mL), the mixture was heated to 80° C. overnight.The reaction mixture was then cooled to rt, and concentrated HCl wasadded to adjust the pH to 5. Precipitate formed was filtered, washedwith water, then dried under high vacuum to yield the product as a whitesolid (205 mg, >99% purity, 94% yield).

¹H NMR (500 MHz, DMSO-d6) δ 10.46 (s, 1H), 7.75 (s, 1H), 6.47 (s, 2H).

LCMS: m/z [M+1]⁺=152.19; R_(T)=0.46 min; purity=>99%.

HPLC conditions: Column: XTerra RP18, 3.5 μm, 3.0×50 mm; Gradient: 5% to100% B in 2.5 minutes; 100% B for 1 minute; 1 mL/min. Eluent A: Milli-QH₂O+0.1% Formic Acid; Eluent B: Acetonitrile+0.1% Formic Acid.

Step Three.6-hydroxy-N-(4-hydroxy-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide(20). To a stirring solution of PA18 (205 mg, 1.36 mmol) in anhydrousDMSO (1.4 mL) was added 1,1′carbonyldiimidazole (331 mg, 2.04 mmol) wasadded at rt, under inert atmosphere. The resulting solution was stirredfor 20 min at rt. In a separate flask containing barbituric acid (174mg. 1.36 mmol) was added anhydrous 1,4-dioxane (4.5 mL), then heated to50° C. Et₃N (303 μL, 2.18 mmol) was added and stirred for 15 min at 50°C. The isocyanate generated from PA18 in DMSO was added to the stirringsuspension, then heated to 80° C. until complete consumption of thestarting materials were observed via LCMS (20 h). The reaction mixturewas cooled to rt, then acidified with 6M HCl (aq), the precipitateformed was isolated, then triturated with H₂O, MeOH, followed byacetonitrile, to yield the product (20) as a brown solid (49 mg, 99.8%purity, 12% yield).

¹H NMR (500 MHz, DMSO-d6+DCl in D₂O) δ 7.29 (s, 1H).

LCMS: m/z [M+1]⁺=306.52; R_(T)=1.61 min; purity=99.8%.

HPLC conditions: Column: XTerra RP18, 3.5 μm, 3.0×50 mm; Gradient: 5% to100% B in 2.5 minutes; 100% B for 1 minute; 1 mL/min. Eluent A: Milli-QH₂O+0.1% Formic Acid; Eluent B: Acetonitrile+0.1% Formic Acid.

Example 9: Preparation of3-((4-(1H-1,2,3-triazol-5-yl)phenyl)amino)-1H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione(21, Formula (VI_(b)), with Reference to FIG. 20)

Compound 17 (70 mg, 0.21 mmol) and HCl (2 mL, 4 N in water) was mixedand reflux for 1 hour, until LCMS showed complete consumption ofstarting material. The precipitate was filtered to yield the product(21) as a white solid (53 mg, 98.9% purity, 87% yield), after dryingunder high vacuum.

¹H NMR (500 MHz, d⁶-DMSO+DCl) δ 8.16 (s, 1H), 7.74 (d, J=8.9 Hz, 2H),7.52 (d, J=8.7 Hz, 2H),

LCMS: m/z [M−1]⁻=309.23; R_(T)=3.23 min; purity=98.9%.

HPLC conditions: Waters XTerra RP18 3.5 μm, 3.0×50 mm; hold 5% B for 1.0minute, 5% to 95% B in 5.0 minutes, then hold 5% B for 1.0 minute, runtime=7.0 min; Eluents: A=0.1% HCO₂H in water; B=0.1% HCO₂H in MeCN.

Example 10: Preparation of6-hydroxy-2,4-dioxo-N-(2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)-1,2,3,4-tetrahydropyrimidine-5-carboxamide(22, Formula (IV_(a)), with Reference to FIG. 21)

To a solution of 5-aminobenzoimidazolone (100 mg, 0.67 mmole) in dryDMSO (1.0 mL) at rt under nitrogen was added CDI (163 mg, 1.01 mmol) inone portion. The reaction mixture was stirred at rt for 2 h. Thisisocyanate intermediate was used as such in the next step.

In a separate flask, to a suspension of barbituric acid (94 mg, 0.74mmol) in anhydrous 1,4-dioxane (1.0 mL) at 55° C. was addedtriethylamine (101 mg, 1.01 mmol) and reaction mixture stirred at 55° C.for 20 min, then the above isocyanate intermediate (solution in DMSO)was added. The resulting reaction mixture was heated at 80° C. for 2 h.Reaction mixture cooled to 0° C., precipitate formed was filtered,washed with water, dried under high vacuum, triturated with ether toprovide the crude product as a light grey powder. Crude product waspurified via ISCO (C18, 0 to 30% acetonitrile in water with ammoniumbicarbonate buffer 10.0 mM over 20 CV gradient) to yield the product(22) as a white solid (96 mg, 99.2% purity, 47% yield).

¹H NMR (500 MHz, DMSO-d6) δ 11.78 (s, 1H), 10.32 (d, J=68.9 Hz, 1H),9.58-9.28 (m, 2H), 7.38 (s, 1H), 7.03 (s, 2H), 6.72 (d, J=16.7 Hz, 2H).

LCMS: m/z [M+1]⁺=304.15; R_(T)=0.89 min; purity=99.2%.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

Example 11: Preparation of3-((4-(1H-1,2,3-triazol-5-yl)phenyl)amino)-5,7-dimethyl-1H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione(23, Formula (VI_(c)), with Reference to FIG. 22)

Step One.5-(bis(methylthio)methylene)-1,3-dimethylpyrimidine-2,4,6(1H,3H,5H)-trione(PA19). To a solution of 1,3-dimethylbarbituric acid (2.00 g, 12.81mmol) and Et₃N (3.75 mL, 26.90 mmol), in DMSO (8.5 mL), was added CS₂(1.55 mL, 25.86 mmol). The resulting solution was stirred 2.5 h at rtbefore addition of Mel (1.60 mL, 25.70 mmol). After 3 h at rt, thereaction was stopped

The reaction was poured into H₂O and extracted twice with AcOEt. Thecombined organic phases were washed with H₂O (×3), dried over MgSO₄,then concentrated to dryness. The residue was triturated with diethylether and the solid was filtrated to provide the desired compound (1.4g, >99% purity). This operation was repeated with the filtrate afterconcentration to yield an additional 1.0 g of product (>99% purity),with a combined mass of 2.4 g (71% yield).

¹H NMR (500 MHz, DMSO) δ 3.19 (s, 6H), 2.56 (s, 6H).

LCMS: m/z [M+1]⁺=261.17; R_(T)=1.15 min; purity=>99%.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

Step Two.5-(((4-(1H-1,2,3-triazol-5-yl)phenyl)amino)(methylthio)methylene)-1,3-dimethylpyrimidine-2,4,6(1H,3H,5H)-trione(PA20). A mixture of PA19 (488 mg, 1.87 mmol) and aniline XX (300 mg,1.87 mmol), in dioxane (12 mL) was heated at 85° C. for 1 h. The solventwas evaporated in vacuo to provide the product (746 mg, 96% yield, 90%purity).

LCMS: m/z [M+1]⁺=373.17; R_(T)=1.22 min; purity=90%.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

Step Three.(E)-N-(4-(1H-1,2,3-triazol-5-yl)phenyl)-1,3-dimethyl-2,4,6-trioxohexahydropyrimidine-5-carbohydrazonamide(PA21).

To a solution of PA21 (125 mg, 0.34 mmol), in dioxane (6 mL), was addedthe hydrazine (0.215 mL, 3.37 mmol). The reaction was heated at 100° C.for 30 min. The solvent was evaporated in vacuo. The crude material wasused in the next step assuming quantitative yield of 119 mg.

LCMS: m/z [M+1]⁺=357.21; R_(T)=1.01 min; purity=85%.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

Step Four.3-((4-(1H-1,2,3-triazol-5-yl)phenyl)amino)-5,7-dimethyl-1H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione(23). PA21 (119 mg, 0.33 mmol), in an aqueous solution of 4 M HCl (3mL), was heated at 100° C. for 1 h. DMSO was added, the precipitate wasand the filtrate was collected and then concentrated to be purified byreverse phase chromatography (C18 column=30 g, 0 to 40% CH₃CN in waterover a 10 min gradient) to provide the product (23) as a beige solid (10mg, 8% yield, >97% purity), after lyophilization.

¹H NMR (500 MHz, DMSO) δ 8.42 (s, 1H), 8.20 (s, 1H), 7.75 (d, J=8.7 Hz,2H), 7.50 (d, J=7.9 Hz, 2H), 3.36 (s, 3H), 3.19 (s, 3H).

LCMS: m/z [M+1]⁺=339.18; R_(T)=1.10 min; purity >97%.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

Example 12: Preparation ofN-(4-(1H-1,2,3-triazol-5-yl)phenyl)-6-hydroxy-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboximidamide(25, Formula (I_(f)), with Reference to FIG. 23)

A solution of PA20 (50 mg, 0.13 mmol), in a solution of NH₃ (1 mL, 7 Min MeOH) was heated at 50° C. for 4 h. The reaction was cooled to rt,the precipitate formed was filtered and collected. The solid was added amixture of CH₃CN—H₂O to provide the title compound (25) (19 mg, 42%yield, >99% purity), after lyophilization.

¹H NMR (500 MHz, DMSO) δ 15.13 (br s, 1H), 12.34 (s, 1H), 10.22 (s, 1H),8.39 (s, 1H), 7.97 (d, J=8.4 Hz, 2H), 7.80 (s, 1H), 7.42 (d, J=8.4 Hz,2H), 3.18 (d, J=8.7 Hz, 6H).

LCMS: m/z [M+1]⁺=342.09; R_(T)=1.22 min; purity >99%.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

Example 13: Preparation ofN-(4-(3H-1,2,3-triazol-4-yl)phenyl)-4-hydroxy-1-methyl-2-(methylthio)-6-oxo-1,6-dihydropyrimidine-5-carboxamide(26, Formula (II_(a)), with Reference to FIG. 24)

Step One. 6-hydroxy-3-methyl-2-(methylthio)pyrimidin-4(3H)-one (PA23). Amixture of N-methyl thiourea (2.00 g, 22.2 mmol), diethyl malonate (3.4mL, 22.2 mmol), and sodium methoxide (10.1 mL, 44.4 mmol, 4.4 M inmethanol) was heated to reflux for 3 h. The reaction was then cooled to50° C., iodomethane (1.4 mL, 22.2 mmol) was added in one-portion andallowed to stir for an additional 30 min at the same temperature. Thesolid formed was filtered, then collected, dissolved in water,neutralized with glacial acetic acid. The precipitate formed wasfiltered and washed with water to yield the product as a white solid(2.62 g, >99% purity, 69% yield).

¹H NMR (500 MHz, DMSO-d6) δ 3.31 (s, 1H), 2.52 (s, 1H).

LCMS: m/z [M+1]⁺=172.84; R_(T)=0.89 min; purity=>99%.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

Step Two.N-(4-(3H-1,2,3-triazol-4-yl)phenyl)-4-hydroxy-1-methyl-2-(methylthio)-6-oxo-1,6-dihydropyrimidine-5-carboxamide(26). Triphosgene (23 mg, 0.0766 mmol) was added to a solution of XX (35mg, 0.219 mmol) and iPr₂NEt (114 μL, 0.657 mmol) in anhydrous THF (730μL), at 0° C., under inert atmosphere. The reaction was allowed to stirat 0° C. for 1 h, anhydrous DMF (1.0 mL) was then added to form ahomogenous solution. In a separate flask containing PA23 (45 mg, 0.263mmol) was dissolved in anhydrous DMF (3.4 mL), the solution was cooledto 0° C. NaH (11 mg, 0.285 mmol, 60% dispersed in oil) was added at 0°C., stirred at the same temperature for 5 min, then allowed to warm upto rt over 30 min. The isocyanate generated from XX in THF/DMF was addeddropwise to the stirring suspension, the reaction was heated to 60° C.for 2 h. The reaction mixture was cooled to rt, the solid formed wasfiltered and collected. The crude product was purified via preparativechromatography (C18, 10 to 100% acetonitrile in water with ammoniumformate buffer 10 mM, over 10 min gradient) to yield the product (26) asa yellow solid (5.5 mg, 96.45 purity, 7% yield), after lyophilization.

¹H NMR (500 MHz, DMSO-d6) δ 15.48 (s, 1H), 11.77 (s, 1H), 7.90 (d, J=8.6Hz, 2H), 7.74 (d, J=8.7 Hz, 2H), 3.49 (s, 3H), 2.63 (s, 3H).

LCMS: m/z [M+1]⁺=359.17; R_(T)=1.49 min; purity=96.4%.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

Example 14: Preparation ofN-(4-(1H-1,2,3-triazol-4-yl)phenyl)-2,4-dihydroxy-1-methyl-6-oxo-1,6-dihydropyrimidine-5-carboxamide(27, Formula (I_(g)), with Reference to FIG. 25)

Step One. 2,6-Dihydroxy-3-methylpyrimidin-4(3H)-one (PA24). To thestirring suspension of N-methyl urea (2.00 g, 27.0 mmol) and diethylmalonate (4.10 mL, 4.32 mmol) in methanol (11 mL) was slowly added NaOMe(12.2 mL, 54.0 mmol, 4.4 M in MeOH). The resulting solution was heatedto reflux and stirred overnight (15 h). The reaction mixture was thencooled to room temperature, and then acidified using an aqueous solutionof HCl (6M). The resulting mixture was concentrated, then added iPrOH,stirred at 40° C. for 30 min. The precipitate was filtered, washed withminimal water, then washed with iPrOH, then dried under high vacuum. Theproduct was obtained as a white solid (4.58 g, 55% purity, 66% yield).

¹H NMR (500 MHz, DMSO-d6) δ 3.59 (s, 2H), 3.06 (s, 3H).

LCMS: m/z [M+1]⁺=143.72; R_(T)=0.22 min; purity=>99%.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

Step Two.N-(4-(1H-1,2,3-triazol-4-yl)phenyl)-2,4-dihydroxy-1-methyl-6-oxo-1,6-dihydropyrimidine-5-carboxamide(27). To a stirring solution of XX (39 mg, 0.251 mmol, 97% purity) inanhydrous DMSO (250 μL) was added 1,1′carbonyldiimidazole (61 mg, 0.377mmol) was added at rt, under inert atmosphere. The resulting solutionwas stirred for 20 min at rt. In a separate flask containing PA24 (71mg, 0.502 mmol) was added anhydrous 1,4-dioxane (840 μL), then heated to50° C. Et₃N (105 mL, 0.753 mmol) was added and stirred for 15 min at 50°C. The isocyanate generated from XX in DMSO was added to the stirringsuspension, then heated to 80° C. until complete consumption of thestarting materials were observed via LCMS. After 30 min, the reactionmixture was cooled to rt, then acidified with 6M HCl (aq), theprecipitate formed was isolated, then triturated with H₂O, MeOH,followed by acetonitrile. The solid was then lyophilized to removetraces of water to yield the product (27) as a white solid (45.5 mg,99.5% purity, 55% yield).

¹H NMR (500 MHz, DMSO-d6) δ 14.97 (s, 1H), 12.33 (s, 1H), 11.70 (s, 1H),8.30 (s, 1H), 7.90 (d, J=8.6 Hz, 2H), 7.65 (d, J=8.7 Hz, 2H), 3.18 (s,3H).

LCMS: m/z [M+1]⁺=329.14; R_(T)=1.18 min; purity=99.5%.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

Example 15: Preparation ofN-(4-(3H-1,2,3-triazol-4-yl)phenyl)-4-hydroxy-2-methoxy-6-oxo-1,6-dihydropyrimidine-5-carboxamide(28, Formula (II_(b)), with Reference to FIG. 26)

To a stirring solution of XX (73 mg, 0.457 mmol, 97% purity) inanhydrous DMSO (460 μL) was added 1,1′carbonyldiimidazole (111 mg, 0.686mmol) was added at rt, under inert atmosphere. The resulting solutionwas stirred for 20 min at rt. In a separate flask containing6-hydroxy-2-methoxypyrimidin-4(3H)-one (65 mg, 0.457 mmol) was addedanhydrous 1,4-dioxane (1.5 mL), then heated to 50° C. Et₃N (102 μL,0.731 mmol) was added and stirred for 15 min at 50° C. The isocyanategenerated from XX in DMSO was added to the stirring suspension, thenheated to 80° C. until complete consumption of the starting materialswere observed via LCMS. After 20 h, the reaction mixture was cooled tort, then acidified with 6M HCl (aq), the precipitate formed wasisolated, then triturated with H₂O, MeOH, followed by acetonitrile. Thecrude product was purified via ISCO (C18, 0 to 100% acetonitrile inwater with ammonium formate buffer 10 mM over 20 CV gradient), afterlyophilization, yield the product (28) as a white solid (4.8 mg, >99%purity, 3% yield).

¹H NMR (500 MHz, DMSO-d6) δ 12.15 (s, 1H), 9.55 (s, 1H), 8.17 (s, 1H),7.69 (d, J=8.6 Hz, 2H), 7.61 (d, J=8.7 Hz, 2H), 3.29 (s, 3H).

LCMS: m/z [M+1]⁺=328.83; R_(T)=1.01 min; purity=>99%

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

Example 16: Preparation of2-(4-(1H-1,2,3-triazol-5-yl)phenyl)-4,6-dihydroxy-1H-pyrazolo[3,4-d]pyrimidin-3(2H)-one-ammoniasalt (29, Formula (VII_(a)), with Reference to FIG. 27)

To a stirring solution of XX (105 mg, 0.656 mmol) in HCl (6 M in H₂O)was added a solution of NaNO₂ (48 mg, 0.689 mmol) in H₂O (0.1 mL) at 0°C. The reaction was kept at the same temperature for 1 h. Tin (II)chloride monohydrate (296 mg, 1.31 mmol) dissolved in HCl (6 M in H₂O)was added dropwise to the reaction mixture at 0° C. The reaction wasthen allowed to warm up to rt over 3 h. The reaction mixture was thenbasified with 2 M NaOH, then extracted with ethyl acetate and a mixtureof 25% isopropanol in chloroform. The aqueous and organic fractions werecombined, then acidified with HCl (6 M in H₂O), the precipitate wasfiltered off, and the filtrate was concentrated to yield the productPA25 in an aqueous solution.

Crude hydrazine salt PA25 aqueous solution was added methyl4,6-dichloropyrimidine-5-carboxylate (90 mg, 0.437 mmol) dissolved inEtOH (2.2 mL). The reaction mixture was added 2 M NaOH (1.1 mL), thensealed in a pressure vessel and heated to 100° C. for 15 h. The reactionwas allowed to cool to rt, then concentrated under reduced pressure. Thecrude product was purified via ISCO (C18, 0 to 100% acetonitrile inwater with ammonium formate buffer 10 mM over 20 CV gradient), afterlyophilization, yield the product (29) as an orange solid (14.7 mg,98.6% purity, 11% yield).

¹H NMR (500 MHz, DMSO-d6) δ 8.34 (s, 1H), 7.87 (dt, J=8.1, 1.6 Hz, 2H),7.49-7.44 (m, 2H), 7.38-7.33 (m, 1H).

HRMS (ESI+) m/z calculated for C₁₃H₁₃N₈O₃ [M+NH₄]⁺: 329.11. found:329.11.

LCMS: m/z [M+1]⁺=312.0; R_(T)=1.17 min; purity=98.6%.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

Example 17: Preparation ofN-(4-(3H-1,2,3-triazol-4-yl)phenyl)-4-hydroxy-2-methoxy-6-oxo-1,6-dihydropyrimidine-5-carboxamide(30, Formula (I_(h)), with Reference to FIG. 28)

Step One.5-(bis(methylthio)methylene)-1-methylpyrimidine-2,4,6(1H,3H,5H)-trione(PA26). A mixture of 6-hydroxy-3-methylpyrimidine-2,4(1H,3H)-dione(PA24) (2.339 g, 7.04 mmol, 55% purity), triethylamine (2.0 mL, 14.1mmol), and carbon disulfide (850 μL, 14.1 mmol) was allowed to stir atrt until complete consumption of PA24 was observed via LCMS (0.5 h). Theresulting reaction mixture was cooled in an ice-bath, iodomethane (880μL, 14.1 mmol) was added in one-portion, the reaction was then allowedto warm up to rt over 20 h. The reaction mixture was poured into aice-water with rapid stirring, then allowed to stand for 6 h. Theprecipitate formed was filtered, washed with water and diethyl ether.The precipitate was collected and dried under high vacuum to yield theproduct as a yellow solid (97 mg, 6% yield).

¹H NMR (500 MHz, DMSO-d6) δ 3.12 (s, 3H), 2.55 (s, 6H).

LCMS: m/z [M+1]⁺=246.93; R_(T)=0.99 min.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

Step Two.(E)-5-((4-(1H-1,2,3-triazol-4-yl)phenylamino)(methylthio)methylene)-1-methylpyrimidine-2,4,6(1H,3H,5H)-trione(PA27). A mixture of5-(bis(methylthio)methylene)-1-methylpyrimidine-2,4,6(1H,3H,5H)-trione(PA26) (22 mg, 0.0894 mmol, 1 eq) and4-(1H-1,2,3-triazol-4-yl)benzenamine (XX) (14 mg, 0.0894 mmol, 1 eq) inanhydrous 1,4-dioxane (2.2 mL, 0.04 M) was sealed in a microwave vesseland heated to 100° C., until complete consumption of the startingmaterial was observed via LCMS (20 h). The reaction mixture was cooledto rt, then concentrated to yield the crude product (PA #) as anoff-white solid. The crude product was used without furtherpurification.

LCMS: m/z [M+1]⁺=358.92; R_(T)=1.06 min; purity=>99%.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

Step Three.N-(4-(3H-1,2,3-triazol-4-yl)phenyl)-4-hydroxy-2-methoxy-6-oxo-1,6-dihydropyrimidine-5-carboxamide(30). The crude product, PA27 was added a solution of 7.0 M NH₃ inmethanol (1.0 mL) at rt. The reaction was sealed and heated to 60° C.,until complete consumption of the starting material was observed viaLCMS (20 h). The reaction mixture was allowed to cool to rt, theprecipitate formed was filtered and washed with acetonitrile, then thesolid was lyophilized to yield the product (30) as an off-white solid(19 mg, 98.8% purity, 66% yield over 2 steps).

¹H NMR (500 MHz, DMSO-d6) δ 12.28 (s, 1H), 10.14 (s, 1H), 8.41 (s, 1H),7.97 (d, J=8.5 Hz, 2H), 7.76 (s, 1H), 7.42 (d, J=8.4 Hz, 2H), 3.12 (s,3H).

LCMS: m/z [M+1]⁺=328.12; R_(T)=1.04 min; purity=98.8%.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

Example 18: Preparation ofN-(2,3-dioxoindolin-5-yl)-6-hydroxy-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide(32, Formula (IV_(b)), with Reference to FIG. 29)

To a suspension of 31 (20.5 mg, 0.0570 mmol) in MeOH/1,4-dioxane (1.1mL, 1:1 v/v, 0.05 M) was added 6 M HCl (110 μL). The resulting reactionmixture was sealed and heated in the microwave at 100° C. for 20 min.Acetonitrile (5 mL) was added, the precipitate formed was filtered,washed with water, and then acetonitrile to yield the product (32) as adark red solid (7.9 mg, 44% yield).

¹H NMR (500 MHz, DMSO-δ6+AcOD) δ 7.68 (d, J=2.1 Hz, 1H), 7.50 (dd,J=8.4, 2.3 Hz, 1H), 6.86 (d, J=8.5 Hz, 1H).

LCMS: m/z [M+1]⁺=317.36; R_(T)=0.88 min; purity=>99%.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

Example 19: Preparation of6-hydroxy-4-oxo-N-(4-(4-phenyl-1H-1,2,3-triazol-5-yl)phenyl)-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide(33, Formula (I_(m)), with Reference to FIG. 30)

Step One. 1-nitro-4-(phenylethynyl)benzene (PA30). To a round-bottomedflask was added Pd(OAc)₂ (21 mg, 0.094 mmol), PPh₃ (96 mg, 0.366 mmol),CuI (19 mg, 0.100 mmol), 4-iodoaniline (2.0 g, 9.13 mmol) and THF (24mL). After bubbling N₂ through the reaction mixture for 5 min,phenylacetylene (1.20 mL, 10.91 mmol) and Et₃N (6.40 mL, 45.91 mmol)were added sequentially. The reaction was stirred overnight at rt. Afterdiluting with sat. NH₄Cl, the mixture was extracted twice with AcOEt,the combined organic extracts were dried over MgSO₄, then concentratedto dryness. The residue was triturated with diethyl ether and the solidwas filtrated and collected. This operation was repeated with thefiltrate after concentration, the solid collected were combined to yieldthe product (0.69 g, 85% purity). The remaining residue was purified bycombi-flash chromatography (dry pack, SiO column=80 g, 5% ethyl acetatein hexanes) to provide the titled compound (0.76 g, 95% purity) of thetitle compound was isolated with a combined mass of 1.45 g (93% yield).

LCMS: R_(T)=1.96 min; purity=95%.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

¹H NMR (500 MHz, CDCl₃) δ 8.23 (d, J=9.0 Hz, 2H), 7.67 (d, J=9.0 Hz,2H), 7.58-7.55 (m, 2H), 7.42-7.37 (m, 3H).

Step Two. 5-(4-nitrophenyl)-4-phenyl-1H-1,2,3-triazole (PA31). To astirred solution of PA30 (690 mg, 3.09 mmol), in DMF (7.5 mL), was addedNaN₃ (230 mg, 3.54 mmol). The resulting solution was heated at 170° C.for 3.5 h. The reaction mixture, diluted with H₂O, was extracted twicewith AcOEt. The combined organic phases were washed with H₂O (×3) andthen brine before it was dried over MgSO₄, then concentrated to drynessto yield the product (0.82 g, 99% yield, 96% yield).

LCMS: m/z [M+1]⁺=267.01; R_(T)=1.58 min; purity=96%.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

Step Three. tert-butyl5-(4-nitrophenyl)-4-phenyl-1H-1,2,3-triazole-1-carboxylate (PA32). To astirred solution of PA31 (400 mg, 1.50 mmol), in CH₂Cl₂ (5 mL), wasadded a solution of Boc₂O (394 mg, 1.80 mmol), in CH₂Cl₂ (5 mL),followed by addition of Et₃N (0.25 mL, 1.78 mmol). The resultingsolution was stirred at rt for 2 h. The solvent was evaporated in vacuo.The residue was purified by combi-flash chromatography (SiO column=40 g,5% ethyl acetate in hexanes) to yield the product (530 mg, 96% yield,97% purity).

LCMS: m/z [M-Boc]⁺=266.94; R_(T)=1.96 min; purity=97%.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

Step Four. tert-butyl5-(4-aminophenyl)-4-phenyl-1H-1,2,3-triazole-1-carboxylate (PA33). Asuspension of PA32 (223 mg, 0.609 mmol), in EtOH (10 mL), was treatedwith Pd/C 10% (100 mg), 15 mL of MeOH was added. The reaction was fittedwith a hydrogen filled balloon and stirred at rt for 2 h. The reactionwas filtered through a Millex syringe filter. Solvents were evaporatedin vacuo to provide the expected amine (190 mg, 92% yield, >95% purity).

LCMS: m/z [M-Boc]⁺=236.99; R_(T)=1.74 min; purity >95%.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

Step Five.6-hydroxy-4-oxo-N-(4-(4-phenyl-1H-1,2,3-triazol-5-yl)phenyl)-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide(33). To a solution of triphosgene (60 mg, 0.202 mmol), in THF (1.0 mL)at 0° C., was added PA33 (190 mg, 0.565 mmol), in THF (2.0 mL), followedby Et₃N (0.120 mL, 0.840 mmol). The ice bath was removed and theresulting suspension was stirred 45 min at rt. To the thiobarbituricacid (90 mg, 0.624 mmol), in dioxane (4.5 mL) was added Et₃N (0.120 mL,0.840 mmol). The resulting mixture was stirred 15 min at 55° C. To thissuspension, was added the previous suspension of isocyanate generated inTHF, followed by DMSO (1.0 mL) and the resulting solution was stirred 1h at 80° C. The reaction was cooled to rt, the solid formed wasfiltered. The filtrate was then diluted with H₂O, the productprecipitated and was filtered to yield the desired product as the Et₃Nsalt (43 mg, 95% purity). The aqueous phase was then extracted twicewith AcOEt. The product started to precipitate in the organic phase. Thetwo phases were separated and AcOEt was evaporated in vacuo. The residuewas triturated in AcOEt and the solid was filtered to yield the product(69 mg, 90% purity). 30 mg of this isolated solid was purified byreverse phase chromatography (C18 column=30 g, 0 to 50% of CH₃CN inwater, over 10 min gradient) to yield 33 (8 mg, 97% purity), afterlyophilization. Note: Deprotection was observed during the purification,only 33 was isolated.

LCMS: m/z [M+1]⁺=407.14; R_(T)=1.24 min; purity=97%.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

¹H NMR (500 MHz, DMSO) δ 12.06 (s, 1H), 11.11 (s, 2H), 7.62 (d, J=8.6Hz, 2H), 7.53-7.48 (m, 2H), 7.44-7.29 (m, 5H).

Example 20: Preparation ofN-(1,1-dioxido-3-oxo-2,3-dihydrobenzo[d]isothiazol-6-yl)-2,4,6-trihydroxypyrimidine-5-carboxamide(34, Formula (III_(a)), with Reference to FIG. 31)

Step One. 6-Aminobenzo[d]isothiazol-3(2H)-one 1,1-dioxide (PA34).6-Nitrobenzo[d]isothiazol-3(2H)-one 1,1-dioxide (500 mg, 2.19 mmol) wasdissolved in THF (11.0 mL). To the solution was added Pd/C (50 mg, 10%on charcoal) under N₂. The reaction was then evacuated and purged withH₂ (5×), then allowed to stir under H₂ for 4 h. After LCMS showed thatthe reaction was completed, the reaction was mixture was filteredthrough a pad a Celite. The filtrate was collected and the solvent wasremoved in vacuo to yield the product as a yellow solid (409 mg, 94%yield), without further purification.

¹H NMR (500 MHz, MeOD) δ 7.61 (d, J=8.5 Hz, 1H), 6.95 (d, J=1.9 Hz, 1H),6.91 (dd, J=8.5, 2.0 Hz, 1H).

LCMS: m/z [M−1]⁻=199.07; R_(T)=0.25 min.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

Step Two.N-(1,1-dioxido-3-oxo-2,3-dihydrobenzo[d]isothiazol-6-yl)-2,4,6-trihydroxypyrimidine-5-carboxamide(34). Compound 34 was synthesized following the General Procedure 1. Toa stirring solution of PA34 (267 mg, 0.135 mmol) in anhydrous DMSO (1.4mL, 1.0 M) was added 1,1′carbonyldiimidazole (328 mg, 2.02 mmol) wasadded at rt, under inert atmosphere. The resulting solution was stirredfor 20 min at rt. In a separate flask containing barbituric acid (173mg, 1.35 mmol) was added anhydrous 1,4-dioxane (4.5 mL, 0.30 M), thenheated to 55° C. Et₃N (300 μL, 2.16 mmol) was added and stirred for 15min at 55° C. The isocyanate generated from the amine in DMSO was addedto the stirring suspension, then heated to 80° C. until completeconsumption of the starting materials were observed via LCMS (2 h). Thereaction mixture was cooled to rt, then acidified with 6M HCl (aq), theprecipitate formed was isolated, then triturated with water, MeOH,followed by acetonitrile. The crude product was further purified byreverse-phase chromatography (C18, gradient eluent from 0 to 30%acetonitrile in water with 10 mM ammonium formate buffer over 20 CV),fractions containing the product were collected and concentrated todryness. The impure solid was then added acetonitrile and sonicated for15 min, the resulting suspension was filtered, and the solid was washedwith additional acetonitrile. The solid was collected and then added 10%H₂O/CH₃CN and sonicated for 30 min. The resulting suspension wasfiltered and the solid was washed with additional acetonitrile, to yieldthe pure product (34) as an off-white solid (113 mg, >99% purity, 24%yield).

¹H NMR (500 MHz, DMSO-δ6+AcOD) δ 8.18 (s, 1H), 7.67 (d, J=8.3 Hz, 1H),7.57-7.52 (dd, J=8.3, 1.8 Hz, 1H).

LCMS: m/z [M−1]⁻=351.1; R_(T)=2.88 min; purity=>99%.

HPLC conditions: Atlantis T3, 3 μm, 4.6×30 mm; Isocratic 2% B for 0.5minute, 2% to 100% B in 6.0 minutes; hold 100% B for 1.0 minute, 100% to2% B in 0.05 minute, hold 2% B for 0.5 min, run time=8.0 min; Flow rate:1 mL/min; Eluents: A=Milli-Q H2O+0.1% Formic acid; B=MeCN.

Example 21: Preparation of6-hydroxy-2,4-dioxo-N-(4-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)phenyl)-1,2,3,4-tetrahydropyrimidine-5-carboxamide(37, Formula (I_(n)), with Reference to FIG. 32)

Step One. 4-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)aniline (PA43).Ammonium chloride (253 mg, 4.73 mmol), in H₂O (5.0 mL) was added to asolution of PA36 (300 mg, 1.16 mmol), in THF (15.0 mL). Zinc powder (305mg, 4.66 mmol) was then added. The reaction was stirred for 4 days at65° C. The reaction was diluted with H₂O, and then extracted twice withAcOEt. The combined organic extract was dried over MgSO₄, and thenconcentrated. The residue was purified by combi-flash chromatography(SiO₂ column=40 g, 20% ethyl acetate in hexanes) to yield the desiredcompound (119 mg, 44% yield, 98% purity).

LCMS: m/z [M+1]⁺=229.93; R_(T)=1.64 min; purity=98%.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

¹H NMR (500 MHz, CDCl₃) δ 7.90 (d, J=8.7 Hz, 2H), 6.74 (d, J=8.7 Hz,2H), 4.07 (br s, 2H).

Step Two.6-hydroxy-2,4-dioxo-N-(4-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)phenyl)-1,2,3,4-tetrahydropyrimidine-5-carboxamide(37). To a solution of triphosgene (20 mg, 0.067 mmol), in THF (0.25 mL)at 0° C., was added PA43 (41 mg, 0.179 mmol), in THF (0.75 mL), followedby Et₃N (0.038 mL, 0.272 mmol). The ice bath was removed and theresulting suspension was stirred 45 min at rt. To the barbituric acid(26 mg, 0.203 mmol) in dioxane (1.5 mL) was added Et₃N (0.038 mL, 0.272mmol). The resulting mixture was stirred 20 min at 55° C. To thissuspension was added the previous suspension of the isocyanate generatedin THF, followed by the addition of 0.5 mL of DMSO and the resultingsolution was stirred 1.5 h at 80° C.

The reaction was filtered to remove the precipitate. Solvents of thefiltrate were evaporated in vacuo and H₂O was added to the residue. Theproduct crashed out and was filtered to provide the desired product asan Et₃N salt (50 mg). This solid was partially dissolved in a mixture ofAcOEt and 1M HCl. The mixture was vigorously stirred for 1 h. Theaqueous and organic layers were separated, and the solid formed in theorganic phase was filtered. The solid was collected, then added CH₃CNand H₂O and lyophilized to provide a solid. The solid was triturated inDMSO to provide the desired product (37) (8 mg, 11% yield, 97% purity),after lyophilization.

¹H NMR (500 MHz, DMSO) δ 11.77 (s, 2H), 8.09 (d, J=7.7 Hz, 2H), 7.80 (d,J=7.8 Hz, 2H).

LCMS: m/z [M−1]⁻=382.13; R_(T)=1.43 min; purity=97%.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

Example 22: Preparation ofN-(1,1-dioxido-3-oxo-2,3-dihydrobenzo[d]isothiazol-6-yl)-2,4,6-trihydroxypyrimidine-5-carboxamide(39, Formula (I_(i)), with Reference to FIG. 33)

Step One. tert-Butyl trans 4-(1H-1,2,3-triazol-5-yl)cyclohexyl)carbamate(PA48). To a stirring solution of tert-butyl trans4-ethynylcyclohexylcarbamate (170 mg, 0.761 mmol) dissolved in MeOH/DMF(1.5 mL, 1:9 v/v) at room temperature, under inert atmosphere was addedCuI (14 mg, 0.0761 mmol) in one-portion, followed by the addition oftrimethylsilyl azide (150 μL, 1.12 mmol). The reaction was sealed in apressure vessel and heated to 100° C. for 20 h, progress of the reactionwas monitored by LCMS. The reaction was then allowed to cool to rt, thenconcentrated. The crude product was purified via ISCO (0 to 50% ethylacetate in hexanes over 15 CV) to yield the product as a white solid(100 mg, 50% yield).

Rf=0.31 (50% ethyl acetate in hexanes).

¹H NMR (500 MHz,) δ 7.81 (d, J=0.9 Hz, 1H), 7.69 (s, 1H), 3.61 (s, 1H),2.93 (tt, J=12.1, 3.3 Hz, 1H), 2.33-2.25 (m, 4H), 1.76 (dddd, J=12.9,12.9, 3.2, 3.2, 2H), 1.54 (dddd, J=13.4, 13.4, 3.6, 3.6 2H).

LCMS: m/z [M+1]⁺=267.13; R_(T)=1.29 min.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 1 mL/min; 3 minrun. Eluent A: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B:Acetonitrile.

Step Two. Trans-4-(1H-1,2,3-Triazol-5-yl)cyclohexanamonium chloride(PA49). tert-Butyl trans-4-(1H-1,2,3-triazol-5-yl)cyclohexyl)carbamatePA48 (100 mg, 0.377 mmol) was dissolved in dichloromethane (0.75 mL) andmethanol (0.75 mL) at rt. A solution of HCl (4.0 M in dioxane) was addedin one-portion and allowed to stir at rt for 20 h. The reaction mixturewas then concentrated to yield the product as a white solid (76 mg, >99%purity, 100% yield), without further purification.

¹H NMR (500 MHz, MeOD) δ 8.35 (s, 1H), 3.20-3.11 (m, 1H), 3.02-2.92 (m,1H), 2.22-2.08 (m, 4H), 1.69-1.50 (m, 4H).

LCMS: R_(T)=0.25 min; purity=>99%.

HPLC conditions: Atlantis T3, 3 μm, 4.6×30 mm; Isocratic 2% B for 0.5minute, 2% to 100% B in 6.0 minutes; hold 100% B for 1.0 minute, 100% to2% B in 0.05 minute, hold 2% B for 0.5 min, run time=8.0 min; Flow rate:1 mL/min; Eluents: A=Milli-Q H2O+0.1% Formic acid; B=MeCN.

Step Three. 6-Aminobenzo[d]isothiazol-3(2H)-one 1,1-dioxide (39). To astirring solution of PA49 (40 mg, 0.198 mmol) in anhydrous DMSO (200 μL,1.0 M) was added Et₃N (55 μL, 0.396 mmol), followed by the addition of1,1′carbonyldiimidazole (48 mg, 0.297 mmol) was added at rt, under inertatmosphere. The resulting solution was stirred for 20 min at rt. In aseparate flask containing barbituric acid (25 mg, 0.198 mmol) was addedanhydrous 1,4-dioxane (660 μL, 0.30 M), then heated to 55° C. Et₃N (44μL, 0.317 mmol) was added and stirred for 15 min at 55° C. Theisocyanate generated from the amine in DMSO was added to the stirringsuspension, then heated to 80° C. until complete consumption of thestarting materials were observed via LCMS (2 h). The reaction mixturewas cooled to rt, then acidified with 6M HCl (aq), the precipitateformed was isolated, then triturated with water, MeOH, followed byacetonitrile. The crude product was further purified by reverse-phasechromatography (C18, gradient eluent from 0 to 100% acetonitrile inwater with 0.1% formic acid buffer over 20 CV) to yield the product(39), after lyophilization, as an off-white solid (12.9 mg, 96.1%purity, 20% yield).

¹H NMR (500 MHz, DMSO-δ6) δ 9.56 (d, J=7.8 Hz, 1H), 8.37-8.32 (m, 1H),8.26 (s, 1H), 7.70 (s, 1H), 7.03 (s, 1H), 5.65 (d, J=7.8 Hz, 1H),2.06-1.94 (m, 4H), 1.54-1.43 (m, 4H).

LCMS: m/z [M+1]⁺=321.2; R_(T)=3.15 min; purity=96.1%.

HPLC conditions: Atlantis T3, 3 μm, 4.6×30 mm; Isocratic 2% B for 0.5minute, 2% to 100% B in 6.0 minutes; hold 100% B for 1.0 minute, 100% to2% B in 0.05 minute, hold 2% B for 0.5 min, run time=8.0 min; Flow rate:1 mL/min; Eluents: A=Milli-Q H2O+0.1% Formic acid; B=MeCN.

Example 23: Preparation of6-hydroxy-N-(4-hydroxy-4-(1H-1,2,3-triazol-4-yl)cyclohexyl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide(40, Formula (I_(j)), with Reference to FIG. 34)

Step One. tert-Butyl (4-ethynyl-4-hydroxycyclohexyl)carbamate (PA50). Asolution of lithium (trimethylsilyl)acetylide (18.6 mL, 0.5 M in THF)was added slowly to tert-butyl (4-oxocyclohexyl)carbamate (991 mg, 4.65mmol) in anhydrous THF (23 mL) at −78° C., under nitrogen. The reactionwas allow to stir at the same temperature for 1 h, then warmed up to rtover 1 h. The resulting reaction mixture was quenched with a saturatedaqueous solution of ammonium chloride, then diluted with ethyl acetate.The layers were separated, and the organic extract was washed with sat.NaHCO₃ (aq), water, and then brine, before drying over MgSO₄, and thenconcentrated to dryness to yield intermediate tert-butyl(4-hydroxy-4-((trimethylsilyl)ethynyl)cyclohexyl)carbamate.

The crude product was dissolved in methanol (15.5 mL), K₂CO₃ (1.94 g,14.1 mmol) was added and allowed to stir at rt for 3 h. The reaction wasthen concentrated, then ethyl acetate was added, the solution was washedwith brine, and the organic extract was dried over MgSO₄, thenconcentrated. The crude product (748 mg) was used without furtherpurification.

Step Two. tert-Butyl(4-hydroxy-4-(1H-1,2,3-triazol-5-yl)cyclohexyl)carbamate (PA51). To astirring solution of PA50 (748 mg, 3.13 mmol) dissolved in MeOH/DMF (6.3mL, 1:9 v/v) at room temperature, under inert atmosphere was added CuI(60 mg, 0.313 mmol) in one-portion, followed by the addition oftrimethylsilyl azide (605 μL, 4.60 mmol). The reaction was sealed in apressure vessel and heated to 100° C. for 20 h, progress of the reactionwas monitored by LCMS. The reaction was then allowed to cool to rt, thenconcentrated. The crude product was purified via ISCO (0 to 100% ethylacetate in hexanes over 15 CV) to yield the product as a brown oil (330mg, 1:1 dr, 37% yield).

LCMS: m/z [M+1]⁺=283.10; R_(T)=1.41 min.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 1 mL/min; 3 minrun. Eluent A: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B:Acetonitrile.

Step Three. 4-Amino-1-(1H-1,2,3-triazol-5-yl)cyclohexanol hydrogenchloride salt (PA52). PA51 (330 mg, 1.18 mmol) was dissolved indichloromethane (2.4 mL) at rt. A solution of HCl (4.2 mL, 4.0 M indioxane) was added in one-portion and allowed to stir at rt for 16 h.The reaction mixture was then concentrated to yield the product as abrown oil (257 mg, 100% yield, 1:1 dr), without further purification.

LCMS: m/z [M+1]⁺=182.88; R_(T)=0.23 min.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 1 mL/min; 3 minrun. Eluent A: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B:Acetonitrile.

Step Four.6-hydroxy-N-(4-hydroxy-4-(1H-1,2,3-triazol-4-yl)cyclohexyl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide(40). To a stirring solution of PA52 (129 mg, 0.590 mmol) in anhydrousDMSO (590 μL, 1.0 M) was added Et₃N (164 μL, 0.1.18 mmol), followed bythe addition of 1,1′carbonyldiimidazole (144 mg, 0.885 mmol) was addedat rt, under inert atmosphere. The resulting solution was stirred for 20min at rt. In a separate flask containing barbituric acid (76 mg, 0.198mmol) was added anhydrous 1,4-dioxane (2.0 mL, 0.30 M), then heated to55° C. Et₃N (130 μL, 0.944 mmol) was added and stirred for 15 min at 55°C. The isocyanate generated from the amine in DMSO was added to thestirring suspension, then heated to 80° C. until complete consumption ofthe starting materials were observed via LCMS (1.5 h). The reactionmixture was cooled to rt, then acidified with 6M HCl (aq), theprecipitate formed was isolated, then triturated with water, MeOH,followed by acetonitrile. The crude product was further purified byreverse-phase chromatography (C18, gradient eluent from 0 to 100%acetonitrile in water with an ammonium formate buffer 10 mM over 20 CV)to yield the product (40), after lyophilization, as a brown solid (4.8mg, 1:1 dr, 2% yield).

¹H NMR (500 MHz, DMSO-δ6) δ (1:1 dr) δ 8.19 (s, 1H), 7.53 (s, 1H), 7.02(s, 1H), 4.47-4.41 (m, 2H), 3.97 (s, 3H), 3.77-3.72 (m, 2H), 3.68-3.65(m, 2H).

Example 24: Preparation ofN-(4-(1H-1,2,3-triazol-5-yl)phenyl)-6-amino-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide(42, Formula (I_(k)), with Reference to FIG. 35)

Triphosgene (342 mg, 1.15 mmol) was added to a solution of XX (527 mg,3.29 mmol) and iPr₂NEt (1.7 mL, 9.87 mmol) in anhydrous THF (11.0 mL),at 0° C., under inert atmosphere. The reaction was allowed to stir at 0°C. for 1 h, anhydrous DMF (1.0 mL) was then added to form a homogenoussolution. In a separate flask containing 6-aminouracil (502 mg, 3.95mmol) was dissolved in anhydrous DMF (32 mL), the solution was cooled to0° C. NaH (171 mg, 4.28 mmol, 60% dispersed in oil) was added at 0° C.,stirred at the same temperature for 5 min, then allowed to warm up to rtover 30 min. The isocyanate generated from XX in THF/DMF was addeddropwise to the stirring suspension, the reaction was heated to 60° C.for 1 h. The reaction mixture was cooled to rt, then concentrated todryness. The crude product was purified via preparative chromatography(C18, 10 to 100% acetonitrile in water with ammonium formate buffer 10mM, over 10 min gradient) to yield the product (42) as an off-whitesolid (32 mg, 98.7 purity, 3% yield), after lyophilization.

¹H NMR (500 MHz, DMSO-d6) δ 8.37 (s, 1H), 7.71 (d, J=8.4 Hz, 2H), 7.55(d, J=8.4 Hz, 2H).

LCMS: m/z [M−1]⁻=312.1; R_(T)=0.98 min; purity=98.7%.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

Example 25: Preparation ofN-(4-(1H-1,2,3-triazol-5-yl)phenyl)-4,6-dihydroxy-2-(methylthio)pyrimidine-5-carboxamide(7, Formula (II_(c)), with Reference to FIG. 36)

2-(Methylthio)pyrimidine-4,6-diol (127 mg, 0.800 mmol) was added to astirring solution of sodium tert-butoxide (78 mg, 0.800 mmol) dissolvedin DMSO (2 mL) at rt for 5 min. In a separate flask, aniline XX wasdissolved in 1,4-dioxane (0.5 mL), to this solution was addedtriphosgene (39 mg, 0.132 mmol) in one-portion. The suspension wasstirred vigorously for 2 min at rt, then iPr₂NEt (139 □L, 0.800 mmol)was added. The suspension was stirred vigorously at rt for 2 min.Freshly prepared solution of sodium6-hydroxy-2-(methylthio)pyrimidin-4-olate in DMSO was added to thesuspension in one-portion. The reaction was stirred at 90° C. for 30min, until complete consumption of starting material observed via LCMS.The reaction mixture was loaded directly on C18 column (60 g) andpurified via ISCO (C18, 0 to 100% acetonitrile in water, with anammonium bicarbonate buffer 10 mM) to yield the product (7) as a whitesolid (60 mg, 44% yield, 97.8% purity), after lyophilisation.

¹H NMR (400 MHz, DMSO-d6+DCl in D₂O) δ 11.71 (s, 1H), 8.32 (s, 1H), 7.86(d, J=8.7 Hz, 2H), 7.68 (d, J=8.6 Hz, 2H), 2.48 (s, 3H).

LCMS: m/z [M−1]⁻=343.0; R_(T)=1.29 min.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 1 mL/min; 3 minrun. Eluent A: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B:Acetonitrile.

Example 26: Preparation of6-hydroxy-N-(4-(hydroxycarbamoyl)phenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide(8, Formula (VIII_(a)), with Reference to FIG. 37)

Step One. Methyl4-(6-hydroxy-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamido)benzoate(PA1). To a stirring solution of methyl 4-aminobenzoate (354 mg, 2.34mmol) in anhydrous DMSO (2.3 mL) was added 1,1′carbonyldiimidazole (569mg, 3.51 mmol) was added at rt, under inert atmosphere. The resultingsolution was stirred for 20 min at rt. In a separate flask containingbarbituric acid (300 mg. 2.34 mmol) was added anhydrous 1,4-dioxane (7.8mL), then heated to 50° C. Et₃N (522 μL, 3.74 mmol) was added andstirred for 15 min at 50° C. The isocyanate generated from methyl4-aminobenzoate in DMSO was added to the stirring suspension, thenheated to 80° C. until complete consumption of the starting materialswere observed via LCMS (2 h). The reaction mixture was cooled to rt,then acidified with 6M HCl (aq), the precipitate formed was isolated,then triturated with MeOH, followed by acetonitrile, to yield theproduct as a off-white solid (342 mg, >99% purity, 48% yield).

¹H NMR (500 MHz, DMSO-d6+AcOD) δ 8.00-7.87 (m, 2H), 7.73-7.64 (m, 2H),3.84 (s, 3H).

LCMS: m/z [M−1]⁻=304.07; R_(T)=2.33 min; purity=>99%.

HPLC conditions: Column: XTerra RP18, 3.5 μm, 3.0×50 mm; Gradient: 5% to100% B in 2.5 minutes; 100% B for 1 minute; 1 mL/min; 4 min run. EluentA: Milli-Q H₂O+0.1% Formic Acid; Eluent B: Acetonitrile+0.1% FormicAcid.

Step Two.6-hydroxy-N-(4-(hydroxycarbamoyl)phenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide(PA2). PA1 (160 mg, 0.525 mmol) was added MeOH/CH₂Cl₂ (4.4 mL, 2:1 v/v)then cooled to 0° C. An aqueous solution of ammonium hydroxide (1.0 mL,15.75 mmol. 50% in water), followed the addition of solid sodiumhydroxide (210 mg, 5.25 mmol). The reaction mixture was then warmed upto rt, sealed and heated to 110° C. in the microwave for 10 min. Aftercooling to rt, the reaction mixture was acidified with 1M HCl (aq) thenconcentrated. The crude product was purified via ISCO (C18, 5 to 100%acetonitrile in water, with an ammonium formate buffer 10 mM, over 15 CVgradient) to yield the product (8) as an off-white solid (48.3 mg, 95.2%purity, 27% yield), after lyophilization.

¹H NMR (500 MHz, DMSO-d6+AcOD) δ 8.08 (br s, 4H).

LCMS: m/z [M−1]⁻=305.12; R_(T)=1.68 min; purity=95.2%.

HPLC conditions: Column: XTerra RP18, 3.5 μm, 3.0×50 mm; Gradient: 5% to100% B in 2.5 minutes; 100% B for 1 minute; 1 mL/min; 4 min run. EluentA: Milli-Q H₂O+0.1% Formic Acid; Eluent B: Acetonitrile+0.1% FormicAcid.

Example 27: Preparation of6-hydroxy-N-(2-(hydroxycarbamoyl)pyridin-4-yl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide(18, Formula (VIII_(b)), with Reference to FIG. 38)

Step One. Methyl 4-aminopicolinate (PA15). To the stirring solution of4-aminopicolinic acid (600 mg, 4.34 mmol) in methanol (31 mL) at 0° C.was slowly added SOCl₂ (3.7 mL). The resulting reaction mixture washeated to reflux for 1 h, then concentrated to yield the titled compoundas a white solid (467 mg, >98% purity, 71% yield) without furtherpurification.

LCMS: m/z [M+1]⁺=152.92; R_(T)=0.48 min; purity=>99%.

HPLC conditions: Column: XTerra RP18, 3.5 μm, 3.0×50 mm; Gradient: 5% to100% B in 2.5 minutes; 100% B for 1 minute; 1 mL/min; 4 min run. EluentA: Milli-Q H₂O+0.1% Formic Acid; Eluent B: Acetonitrile+0.1% FormicAcid.

Step Two. Methyl4-(6-hydroxy-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamido)picolinate(PA16). PA16 was synthesized following general procedure 1. To astirring solution of methyl 4-aminopicolinate PA15 (200 mg, 1.32 mmol)in anhydrous DMSO (1.3 mL) was added 1,1′carbonyldiimidazole (320 mg,1.97 mmol) was added at rt, under inert atmosphere. The resultingsolution was stirred for 20 min at rt. In a separate flask containingbarbituric acid (169 mg. 1.32 mmol) was added anhydrous 1,4-dioxane (4.4mL), then heated to 50° C. Et₃N (294 μL, 2.11 mmol) was added andstirred for 15 min at 50° C. The isocyanate generated from methyl4-aminobenzoate in DMSO was added to the stirring suspension, thenheated to 80° C. until complete consumption of the starting materialswere observed via LCMS (1 h). The reaction mixture was cooled to rt,then acidified with 6M HCl (aq), the precipitate formed was isolated,then triturated with MeOH, followed by acetonitrile, to yield theproduct as an off-white solid (218 mg, >99% purity, 54% yield).

¹H NMR (500 MHz, DMSO-d6) δ 12.48 (s, 1H), 8.88 (s, 1H), 8.41 (d, J=5.4Hz, 1H), 8.26 ((d, J=1.8 Hz, 1H), 7.63 (ddc, J=5.6, 1.7 Hz, 1H), 7.59(s, 2H), 3.86 (s, 3H).

LCMS: m/z [M+1]⁺=307.17; R_(T)=1.48 min; purity=>99%.

HPLC conditions: Column: XTerra RP18, 3.5 μm, 3.0×50 mm; Gradient: 5% to100% B in 2.5 minutes; 100% B for 1 minute; 1 mL/min; 4 min run. EluentA: Milli-Q H₂O+0.1% Formic Acid; Eluent B: Acetonitrile+0.1% FormicAcid.

Step Three.6-hydroxy-N-(2-(hydroxycarbamoyl)pyridin-4-yl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide(18). PA16 (134 mg, 0.438 mmol) was added MeOH/CH₂Cl₂ (3.7 mL, 2:1 v/v)then cooled to 0° C. An aqueous solution of ammonium hydroxide (2.6 mL,15.75 mmol. 50% in water), followed the addition of solid sodiumhydroxide (175 mg, 4.38 mmol). The reaction mixture was then warmed upto rt, sealed and heated to 110° C. in the microwave for 20 min. Aftercooling to rt, the reaction mixture was acidified with 1M HCl (aq) thenconcentrated. The crude product was purified via ISCO (C18, 5 to 100%acetonitrile in water, with an ammonium formate buffer 10 mM, over 15 CVgradient) to yield the product (18) as an off-white solid (7.8 mg, 97.8%purity, 6% yield), after lyophilization.

¹H NMR (500 MHz, DMSO-d6+DCl in D₂O) δ 8.59 (d, J=6.7 Hz, 1H), 8.33 (d,J=1.7 Hz, 1H), 8.20 (d, J=6.7 Hz, 1H).

LCMS: m/z [M+1]⁺=308.52; R_(T)=0.19 min; purity=>99%.

HPLC conditions: XBridge C18, 3.5 μm, 4.6×30 mm; Iso 5% B for 0.2 min,5% to 100% B in 1.8 minutes; hold 100% B for 1 minute, run time=3.0 min;Flow=3 mL/min; Eluents: A=−Q H2O+10 mM Ammonium Bicarbonate pH: 10;B=MeCN.

Example 28: Preparation of2,4,6-trihydroxy-N-(2′-oxospiro[[1,3]dioxolane-2,3′-indolin]-6′-yl)pyrimidine-5-carboxamide(31, Formula (IV_(c)), with Reference to FIG. 39)

Step One. 5′-Nitrospiro[[1,3]dioxolane-2,3′-indolin]-2′-one (PA28). Asolution of 5-aminoisatin (500 mg, 2.60 mmol) and ethylene glycol (290μL, 5.20 mmol) in toluene (5.2 mL, 0.5 M) was added p-toluenesulfonicacid monohydrate (25 mg, 0.13 mmol). The reaction mixture was heated to100° C. equipped with a Dean-Stark apparatus for 20 h, until consumptionof starting material was observed by LCMS. The reaction was then allowedto cool to room temperature; the precipitate formed was collected byvacuum filtration, washed with toluene (10 mL), then dried under highvacuum to yield the product as a brown solid (573 mg, 93% yield).

LCMS: m/z [M+1]⁺=237.05; R_(T)=1.24 min; purity=>99%.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

Step Two. 5′-Aminospiro[[1,3]dioxolane-2,3′-indolin]-2′-one (PA29). PA28(466 mg, 1.97 mmol) dissolved in MeOH (39 mL, 0.05 M) was addedpalladium on charcoal (500 mg, 10 wt. %), the reaction was evacuated andpurged with hydrogen gas (5×), then allowed to stir under hydrogen (1atm, balloon) at room temperature, until complete consumption ofstarting material was observed by LCMS. The reaction mixture was thenfiltered through a small pad of celite, washed with methanol (20 mL),then the filtrate was collected and concentrated to yield the product asa brown solid (406 mg, 100% yield) without further purification.

¹H NMR (500 MHz, MeOD) δ 6.79 (d, J=2.2 Hz, 1H), 6.72 (dd, J=8.2, 2.3Hz, 1H), 6.64 (d, J=8.2 Hz, 1H), 4.49-4.41 (m, 2H), 4.31-4.23 (m, 2H).

LCMS: m/z [M+1]⁺=207.12; R_(T)=0.33 min; purity=>99%.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

Step Three.6-Hydroxy-2,4-dioxo-N-(2′-oxospiro[[1,3]dioxolane-2,3′-indolin]-5′-yl)-1,2,3,4-tetrahydropyrimidine-5-carboxamide(31). Compound 31 was synthesized following the General Procedure 1. Toa stirring solution of aminospiro[[1,3]dioxolane-2,3′-indolin]-2′-one(PA29) (87 mg, 0.422 mmol) in anhydrous DMSO (420 μL, 1.0 M) was added1,1′carbonyldiimidazole (103 mg, 0.633 mmol) was added at rt, underinert atmosphere. The resulting solution was stirred for 20 min at rt.In a separate flask containing barbituric acid (54 mg, 0.422 mmol) wasadded anhydrous 1,4-dioxane (1.4 mL, 0.30 M), then heated to 55° C. Et₃N(94 μL, 0.675 mmol) was added and stirred for 15 min at 55° C. Theisocyanate generated from the amine in DMSO was added to the stirringsuspension, then heated to 80° C. until complete consumption of thestarting materials were observed via LCMS (2 h). The reaction mixturewas cooled to rt, then acidified with 6M HCl (aq), the precipitateformed was isolated, then triturated with water, MeOH, followed byacetonitrile. The crude product was further purified by reverse-phasechromatography (C18, gradient eluent from 0 to 100% acetonitrile inwater with 10 mM ammonium formate buffer over 20 CV) to yield theproduct (31) as a pinkish-brown solid (52 mg, 34% yield), afterlyophilization.

¹H NMR (500 MHz, DMSO-δ6) δ 11.99 (s, 1H), 9.58 (s, 1H), 7.73 (s, 1H),7.27 (d, J=7.6 Hz, 1H), 6.70 (d, J=8.3 Hz, 1H), 4.34 (dd, J=8.2, 4.9 Hz,2H), 4.25 (dd, J=8.5, 5.1 Hz, 2H).

LCMS: m/z [M−1]⁻=359.06; R_(T)=0.97 min; purity=96.1%.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

Example 29: Preparation of4-hydroxy-N-(4-(4-methyl-1H-1,2,3-triazol-5-yl)phenyl)-2-(methylthio)-6-oxo-1,6-dihydropyrimidine-5-carboxamide(43, Formula (II_(g)), with Reference to FIG. 40)

Step One. 1-Nitro-4-(prop-1-yn-1-yl)benzene (PA58). A round bottom flaskcontaining 1-bromo-4-nitrobenzene (1.00 g, 4.95 mmol), PdCl₂(PPh₃)₂(174mg, 0.248 mmol), and CuI (47 mg, 0.248 mmol) was purged with nitrogenfor 15 min. Anhydrous acetonitrile (2.5 mL) was added, followed bypropyne in heptane (13.2 mL, 99.0 mmol, 3% in heptane) and Et₃N (1.4 mL,9.90 mmol). The reaction mixture was sealed and allow to stir at rt for20 h. The reaction mixture was then concentrated, diethyl ether wasadded, then filtered through a small pad of Celite. The filtrate wasconcentrated then purified via ISCO (SiO₂, gradient eluent from 0 to 25%ethyl acetate in hexanes over 20 CV) to yield the product as a yellowsolid (645 mg, >99% purity, 81% yield).

R_(f): 0.79 (25% ethyl acetate in hexanes).

LCMS: R_(T)=1.73 min; purity=>99%.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

Step Two. 5-Methyl-4-(4-nitrophenyl)-1H-1,2,3-triazole (PA59). Sodiumazide (111 mg, 1.71 mmol) was added to PA58 (229 mg, 1.42 mmol)dissolved in anhydrous DMF (7.1 mL) at rt. The reaction was sealed in apressure vessel and heated to 120° C. for 18 h. The reaction mixture wasthen allowed to warm up to rt, dichloromethane was added, followed bywater. The aqueous layer was extracted with dichloromethane (3×20 mL),the combined organic extract was washed with brine, dried over MgSO₄,then concentrated under reduced pressure to yield the product as a brownsolid (180 mg, >99% purity, 62% yield), without further purification.

LCMS: m/z [M+1]⁺=205.29; R_(T)=1.29 min; purity=>99%.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

Step Three. 4-(5-Methyl-1H-1,2,3-triazol-4-yl)aniline (PA60). Tin (II)chloride (938 mg, 4.51 mmol) was added to PA59 (230 mg, 1.13 mmol) inEtOH (3.8 mL) and conc. HCl (710 μL) at rt, the resulting reactionmixture was heated to reflux for 1 h. After complete consumption of thestarting material was observed via LCMS, the reaction was allowed tocool to rt, before pouring into a solution of K₃PO₄ (˜1.0 g) in MeOH (10mL) at rt. The resulting reaction was stirred at rt for 30 min until thepH is not longer acidic. The precipitate was filtered, washed withadditional methanol. The filtrate was collected and concentrated underreduced pressure. The crude product was purified via ISCO (SiO₂,gradient eluent from 0 to 15% methanol in dichloromethane over 12 CV) toyield the product as a brown oil (69 mg, 35% yield).

LCMS: m/z [M+1]⁺=175.42; R_(T)=0.83 min.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

Step Four.4-hydroxy-N-(4-(4-methyl-1H-1,2,3-triazol-5-yl)phenyl)-2-(methylthio)-6-oxo-1,6-dihydropyrimidine-5-carboxamide(43). 43 was synthesized following general procedure 2.2-(Methylthio)pyrimidine-4,6-diol (171 mg, 1.08 mmol) was added to astirring solution of sodium tert-butoxide (104 mg, 1.08 mmol) dissolvedin DMSO (2.7 mL) at rt for 5 min. In a separate flask, aniline PA60 wasdissolved in 1,4-dioxane (680 mL), to this solution was addedtriphosgene (53 mg, 0.178 mmol) in one-portion. The suspension wasstirred vigorously for 2 min at rt, then iPr₂NEt (190 μL) was added. Thesuspension was stirred vigorously at rt for 2 min. Freshly preparedsolution of sodium 6-hydroxy-2-(methylthio)pyrimidin-4-olate in DMSO wasadded to the suspension in one-portion. The reaction was stirred at 90°C. for 30 min, until complete consumption of starting material observedvia LCMS. The reaction mixture was loaded directly on C18 column andpurified via reverse-phase chromatography (gradient eluent from 0 to100% acetonitrile in water with an ammonium formate buffer 10 mM over 20CV) to yield the product (43) as brown solid (29.2 mg, 97.7% purity, 15%yield), after lyophilization.

¹H NMR (400 MHz, DMSO-d6) δ 7.69 (br s, 4H), 2.44 (s, 3H), 2.37 (s, 3H).

LCMS: m/z [M+1]⁺=359.0; R_(T)=1.37 min; purity=97.7%.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

Example 30: Preparation ofN-(4-(1H-1,2,3-triazol-5-yl)phenyl)-4-hydroxy-2-(isopropylthio)-6-oxo-1,6-dihydropyrimidine-5-carboxamide(44, Formula (II_(d)), with Reference to FIG. 41)

Step One. 1-(3,4,5-trimethoxybenzyl)thiourea (PA61).(3,4,5-Trimethoxyphenyl)methanamine (2.5 mL, 14.6 mmol) was addeddropwise to a solution of 1,1′-thiocarbonyl diimidazole (3.91 g, 22.0mmol) dissolved in dichloromethane (36.5 mL) at 0° C. The reactionmixture was then allowed to warm up to rt over 2 h. After completeconsumption of the starting material was observed via LCMS, a solutionof ammonia in methanol (7.5 mL, 52.6 mmol, 7.0 M in MeOH) was added,then stirred for an additional 20 h. The reaction mixture wasconcentrated under reduced pressure, dichloromethane was added, theprecipitate was isolated and washed with additional CH₂Cl₂, then driedunder high vacuum to yield the product as a light pink solid (2.83 g,76% yield).

LCMS: m/z [M+1]⁺=257.07; R_(T)=1.06 min.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

Step Two.6-hydroxy-2-(isopropylthio)-3-(3,4,5-trimethoxybenzyl)pyrimidin-4(3H)-one(PA62). A mixture of PA61 (781 mg, 3.05 mmol), diethyl malonate (465 μL,3.05 mmol), and NaOMe (1.4 mL, 6.10 mmol, 4.4 M in MeOH) in methanol(2.4 mL) was heated to reflux for 3 h. The reaction was then cooled to˜50° C., isopropyl iodide (3.5 mL, 30.5 mmol) was then added inone-portion. The reaction was stirred for an additional 30 min at 50° C.The reaction mixture was then cooled to rt, then concentrated underreduced pressure. The crude product was purified via reverse-phasechromatography (gradient eluent from 0 to 100% acetonitrile in waterwith an ammonium formate buffer 10 mM over 15 CV) to yield the productas a white solid (433 mg, 97.7% purity, 38% yield), afterlyophilization.

LCMS: m/z [M+1]⁺=367.02; R_(T)=1.41 min; purity=97.7%.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

Step Three.N-(4-(1H-1,2,3-triazol-5-yl)phenyl)-4-hydroxy-2-(isopropylthio)-6-oxo-1-(3,4,5-trimethoxybenzyl)-1,6-dihydropyrimidine-5-carboxamide(PA63). PA63 was synthesized following general procedure 2. PA62 (97 mg,0.265 mmol) was added to a stirring solution of sodium tert-butoxide (25mg, 0.265 mmol) dissolved in DMSO (870 μL) at rt for 5 min. In aseparate flask, aniline XX was dissolved in 1,4-dioxane (220 μL), tothis solution was added triphosgene (17 mg, 0.0578 mmol) in one-portion.The suspension was stirred vigorously for 2 min at rt, then iPr₂NEt (60μL) was added. The suspension was stirred vigorously at rt for 2 min.Freshly prepared solution of sodium6-hydroxy-2-(isopropylthio)-3-(3,4,5-trimethoxybenzyl)pyrimidin-4(3H)-olatein DMSO was added to the suspension in one-portion. The reaction wasstirred at 90° C. for 30 min, until complete consumption of startingmaterial observed via LCMS. The reaction mixture was loaded directly onC18 column and purified via reverse-phase chromatography (gradienteluent from 30 to 100% acetonitrile in water with an ammonium formatebuffer 10 mM over 15 CV) to yield the product as brown solid (31.2 mg,80.2% purity, 26% yield), after lyophilization.

LCMS: m/z [M+1]⁺=552.9; R_(T)=1.80 min; purity=80.2%.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

Step Four.N-(4-(1H-1,2,3-triazol-5-yl)phenyl)-4-hydroxy-2-(isopropylthio)-6-oxo-1,6-dihydropyrimidine-5-carboxamide(44). A solution of PA63 (31.2 mg, 0.0452 mmol, 80% purity) indichloromethane (1.5 mL) was added trifluoroacetic acid (270 μL). Theresulting reaction mixture was sealed in a pressure vessel then heatedto 60° C. for 20 h. The reaction mixture was allowed to cool to rt, thenconcentrated under reduced pressure. The crude product was co-evaporatedseveral times with methanol (3×), then purified via reverse-phasechromatography (C18, gradient eluent from 30 to 100% acetonitrile inwater with an ammonium formate buffer 10 mM over 20 CV) to yield theproduct (44) as an off-white solid (6.0 mg, 98.6% purity, 35% yield),after lyophilization.

¹H NMR (400 MHz, DMSO-d6+AcOD) δ 8.23 (s, 1H), 7.87 (d, J=8.2 Hz, 2H),7.68 (d, J=8.2 Hz, 2H), 3.92 (dt, J=13.7, 6.9 Hz, 1H), 1.36 (d, J=6.9Hz, 6H).

LCMS: m/z [M+1]⁺=373.1; R_(T)=1.49 min; purity=98.6%.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

Example 31: Preparation ofN-(5-(1H-1,2,3-triazol-5-yl)pyridin-2-yl)-4,6-dihydroxy-2-(methylthio)pyrimidine-5-carboxamide(45, Formula (II_(e)), with Reference to FIG. 42)

Step One. 5-((Trimethylsilyl)ethynyl)pyridin-2-amine (PA64). To a sealedtube was added 2-amino-5-bromopyridine (1.00 g, 5.8 mmol), Pd(dba)₂Cl₂(202 mg, 0.29 mmol), PPh₃ (151 mg, 0.58 mmol), CuI (110 mg, 0.578 mmol),Et₃N (10 mL) and TMS-acetylene (963 mg, 9.8 mmol) sequentially. Themixture was degassed and heated at 85° C. for 2 h. After completeconsumption of starting material was observed via LCMS, the solvent wasremoved in vacuo and the crude was purified over silica (gradient eluentfrom 0 to 100% ethyl acetate in hexanes). PA64 was obtained as beigesolid (812 mg, 74% yield).

LCMS: m/z [M+1]⁺=191.3; R_(T)=1.55 min

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

Step Two. 5-Ethynylpyridin-2-amine (PA65). PA64 (500 mg, 2.6 mmol) wasdissolved in THF (5 mL) and to this solution was added TBAF (5 mL, 1 Min THF). The reaction was stirred at rt for 10 min and THF was removedin vacuo. The crude was dissolved in EtOAc and this solution was passedthrough a pad of silica and washed with EtOAc. The filtrate wasconcentrated to yield PA65 as beige solid (256 mg, 82% yield).

LCMS: m/z [M+1]⁺=118.8; R_(T)=0.41 min.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

Step Three.N-(5-Ethynylpyridin-2-yl)-4,6-dihydroxy-2-(methylthio)pyrimidine-5-carboxamide(PA66). t-BuONa (136 mg, 1.4 mmol) was dissolved in DMSO (2 mL) and tothis solution was added 2-(methylthio)pyrimidine-4,6-diol (224 mg, 1.4mmol). The solution was stirred at rt for 5 min and left aside for thesecond step. At the same time, PA65 (84 mg, 0.71 mmol) was dissolved inDCE (1 mL) and to the solution was added CDI (115 mg, 0.71 mmol) inone-portion. The suspension was stirred vigorously for 2 min at rt andiPr₂NEt (250 uL, 1.4 mmol) was added. The solution was stirred at rtvigorously for 2 min. Freshly prepared solution of sodium6-hydroxy-2-(methylthio)pyrimidin-4-olate in DMSO was added to thesuspension. The reaction was stirred at 90° C. for 30 min. DCE solventwas removed in vacuo and the product was isolated by ISCO (120 g C18column, gradient eluent from 0 to 50% acetonitrile in water with anammonium bicarbonate buffer 10 mM over 20 CV). Product elutes at 35%MeCN in water. The product was isolated as a beige solid (62 mg, 29%yield), after lyophilization.

LCMS: m/z [M+1]⁺=303.0; R_(T)=1.59 min

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

Step Four.N-(5-(1H-1,2,3-triazol-5-yl)pyridin-2-yl)-4,6-dihydroxy-2-(methylthio)pyrimidine-5-carboxamide(45). PA66 (62 mg, 0.21 mmol) was dissolved in DMSO (2 mL) and to thissolution was added NaN₃ (67 mg, 1.0 mmol). The mixture was stirred at180° C. for 30 min. The product was purified by ISCO (60 g C18 column,gradient eluent from 0 to 50% acetonitrile in water with an ammoniumbicarbonate buffer 10 mM over 20 CV, product elutes at 22% MeCN inwater). The product (45) was isolated as an off-white solid (25 mg, 35%yield), after lyophilization.

1HNMR (500 MHz, DMSO-d6, DCl) δ 12.11 (s, 1H), 8.88 (dd, J=2.4, 0.8 Hz,1H), 8.45 (s, 1H), 8.33 (dd, J=8.6, 2.4 Hz, 1H), 8.22 (dd, J=8.7, 0.8Hz, 1H), 2.56 (s, 3H).

LCMS: m/z [M−1]⁻=346.0; R_(T)=1.29 min; purity=94.4%.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

Example 32: Preparation of4-hydroxy-2-methoxy-N-(4-(4-methyl-1H-1,2,3-triazol-5-yl)phenyl)-6-oxo-1,6-dihydropyrimidine-5-carboxamide(46, Formula (II_(h)), with Reference to FIG. 43)

46 was synthesized following general procedure 1. To a stirring solutionof PA60 (23 mg, 0.132 mmol) in anhydrous DMSO (130 μL) was added1,1′carbonyldiimidazole (33 mg, 0.198 mmol) was added at rt, under inertatmosphere. The resulting solution was stirred for 20 min at rt. In aseparate flask containing 2-methoxypyrimidine-4,6-diol (21 mg, 0.145mmol) was added anhydrous 1,4-dioxane (440 μL), then heated to 50° C.Et₃N (29 μL, 0.211 mmol) was added and stirred for 15 min at 50° C. Theisocyanate generated from the amine in DMSO was added to the stirringsuspension, then heated to 80° C. until complete consumption of thestarting materials were observed via LCMS (30 min). The reaction mixturewas cooled to rt, then acidified with 6M HCl (aq), the reaction mixturewas directly loaded onto a C18 column and purified via ISCO (gradienteluent from 0 to 50% acetonitrile in water with an ammonium bicarbonatebuffer 10 mM over 20 CV) to yield the product (46) as an off-white solid(1.6 mg, 97.0% purity, 4% yield), after lyophilization.

¹H NMR (400 MHz, DMSO-d6) δ 8.14 (s, 1H), 7.69 (s, 4H), 3.79 (s, 3H),2.44 (s, 3H).

LCMS: m/z [M+1]⁺=342.7; R_(T)=1.24 min; purity=97.0%.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

Example 33: Preparation ofN-(3-(1H-1,2,3-triazol-4-yl)bicyclo[1.1.1]pentan-1-yl)-2,4-dihydroxy-6-oxo-1,6-dihydropyrimidine-5-carboxamide(47, Formula (II_(f)), with Reference to FIG. 44)

Step One. tert-Butyl(3-(hydroxymethyl)bicyclo[1.1.1]pentan-1-yl)carbamate (PA67).3-((Tert-butoxycarbonyl)amino)bicyclo[1.1.1]pentane-1-carboxylic acid(600 mg, 2.6 mmol) was added to THF (25 mL). The solution was cooled to0° C. under N₂. To the solution was added LiAH₄ (401 mg, 10.6 mmol)under N₂. The reaction was warmed up to rt and stirred for 1 h. Na₂SO₄decahydrate (500 mg) was added slowly to the reaction and the reactionwas diluted with EtOAc (30 mL). The precipitate was filtered and thefiltrate was concentrated in vacuo to yield crude PA67 (420 mg, 75%yield), which was used without purification.

1HNMR (500 MHz, CDCl₃) δ 3.7 (s, 2H), 1.94 (s, 6H), 1.44 (s, 9H).

Step Two. tert-Butyl (3-formylbicyclo[1.1.1]pentan-1-yl)carbamate(PA68). SO₃—Pyridine (500 mg, 3.1 mmol) was added portion wise (smallexotherm) to a solution of DMSO (1.2 g, 15 mmol), PA67 (335 mg, 1.6mmol) and iPr₂Net (811 mg, 6.3 mmol) in CH₂Cl₂ (6 mL) at rt. Thereaction was stirred for 20 min at rt. The reaction mixture was dilutedwith CH₂Cl₂ (30 mL). The reaction was then washed with sat. NaHCO₃(10mL), brine (10 mL) and dried over MgSO₄ and concentrated in vacuo. Thecrude PA68 was used in the next step, without further purification.

1HNMR (500 MHz, CDCl₃) δ 9.66 (s, 1H), 2.29 (s, 6H), 1.44 (s, 9H).

Step Three. tert-butyl (3-ethynylbicyclo[1.1.1]pentan-1-yl)carbamate(PA69). PA68 (80 mg, 0.38 mmol) was dissolved in dry MeOH/THF (2 mL, 1:1v/v, dried over MgSO₄ overnight). To the solution was added K₂CO₃ (105mg, 0.76 mmol) and dimethyl diazo-2-oxopropylphosphonate (95 mg, 0.49mmol). The mixture was stirred overnight. ISCO purification wasperformed (dry loading with silica, gradient eluent from 0 to 50% ethylacetate in hexanes) to yield the desired product, PA69 (42 mg, 53%yield).

1HNMR (500 MHz, CDCl₃) δ 2.29 (s, 6H), 2.10 (s, 1H), 1.25 (s, 9H).

Step Four. tert-butyl(3-(1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)bicyclo[1.1.1]pentan-1-yl)carbamate(PA70). CuSO₄ (263 mg in 3 mL water, 1.6 mmol) was added to sodiumascorbate (390 mg in 3 mL water, 2.0 mmol). The solution was stirred atrt for 1 min and DMSO (8 mL) was added to the mixture. The suspensionwas added to PA69 (70 mg, 0.33 mmol) and PMB-N₃ (161 mg, 0.99 mmol)mixture in 4 mL MeOH. The resulting mixture was stirred at rt for 30min. The precipitate was filtered by Celite and washed with methanol.The filtrate was concentrated to remove MeOH and the product wasextracted with EtOAc/H₂O (40 mL/20 mL). The organic layer was washedwith brine and dried over MgSO₄, then concentrated in vacuo to give thecrude product. The crude product was purified by silica pad, (30% ethylacetate in hexanes to remove the excess azide. The product was flushedout by 1/1 MeOH/DCM) to yield the desired product (115 mg, 95% yield).

LCMS: m/z [M+1]⁺=371.1; R_(T)=1.61 min

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

Step Five.3-(1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)bicyclo[1.1.1]pentan-1-amine(PA71). PA70 (80 mg, 0.22 mol) was dissolved in TFA (4 mL) and thereaction was stirred at rt for 30 min. Removal of the solvent in vacuoand the crude was dissolved in EtOAc (30 mL). The organic layer waswashed with sat. aqueous Na₂CO₃ (10 mL) and brine (10 mL). The organiclayer was then dried over MgSO₄ and the solvent was removed in vacuo toyield the crude product (58 mg, 99% yield).

LCMS: m/z [M+1]⁺=271.0; R_(T)=1.08 min

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

Step Six.4-Hydroxy-N-(3-(1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)bicyclo[1.1.1]pentan-1-yl)-2-(methylthio)-6-oxo-1,6-dihydropyrimidine-5-carboxamide(PA72). PA71 (57 mg, 0.21 mmol) was dissolved in dioxane (0.3 mL). Tothis solution was added CDI (45 mg, 0.27 mmol), the reaction was stirredat rt for 3 min and iPr₂NEt (82 mg, 0.63 mmol) was added. The solutionwas stirred at rt for 10 min. To this solution was added freshlyprepared 2-(methylthio)pyrimidine-4,6-diol (100 mg, 0.63 mmol) withNaOt-Bu (61 mg, 0.63 mmol) in DMSO (1 mL). The mixture was heated up to90° C. for 1 h until complete consumption of the starting material wasobserved by LCMS. The crude product was then purified by ISCO (gradienteluent from 0 to 100% acetonitrile in water with an ammonium bicarbonatebuffer 10 mM, product eluted at 35% MeCN in water) to yield the product(42 mg, 44% yield), after lyophilization.

LCMS: m/z [M+1]⁺=455.1; R_(T)=1.54 min

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile

Step Seven.N-(3-(1H-1,2,3-Triazol-4-yl)bicyclo[1.1.1]pentan-1-yl)-2,4-dihydroxy-6-oxo-1,6-dihydropyrimidine-5-carboxamide(47). PA72 (20 mg, 0.044 mmol) was dissolved in TFA (4 mL) and TfOH (0.2mL) was added. The reaction mixture was heated at 85° C. for 4 h. To thereaction was added 0.3 mL iPr₂NEt (To prevent decomposition caused byTfOH) and the reaction was concentrated in vacuo. The crude product waspurified by ISCO (gradient eluent from 0 to 100% acetonitrile in waterwith an ammonium bicarbonate buffer 10 mM over 15 CV) to yield theproduct (47) as a white solid (27 mg, 87% yield).

1HNMR (500 MHz, DMSO-d6, TFA) δ 9.91 (s, 1H), 7.74 (s, 1H), 2.55 (s,3H), 2.45 (s, 6H).

LCMS: m/z [M+1]⁺=334.9; R_(T)=1.23 min; purity=97.0%.

HPLC conditions: Column: XBridge C18, 3.5 μm, 4.6×30 mm; Gradient: 5% Bfor 0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min. EluentA: Milli-Q H₂O+10 mM ammonium formate pH: 3.8; Eluent B: Acetonitrile.

Example 34: Bioactivity Assays

The biological activities of compounds having structures represented byany of Formulae (I)-(VII), were evaluated in two assays: xanthineoxidase activity and URAT1 activity.

Xanthine oxidase inhibition was determined using a standardfluorescence-based assay for xanthine oxidase activity (McHale A, GrimesH, Coughlan M P: Int J Biochem. 10:317-9, 1979) with minor variations.The procedure was internally standardized using allopurinol and DPI ascontrols for all experiments after determination of their optimalinhibitory concentrations. Experiments on test compounds were performedin triplicate in multi-well plates using 10 concentrations of eachcompound that ranged over a 3-fold dilution.

URAT1 (SLC22A12) activity was evaluated in a cellular uptake assay usinga 96-well plate with stably transfected URAT-1/CHO cells. ³H-orotate wasused as the test transport agent, which was measured in a liquidscintillation counter, using benzbromarone as a positive control, andDMSO and non-transfected CHO cells as negative controls (SolvoBiotechnology, Boston, Mass.). Generally determined over 7concentrations (range, 0.01 to 150 μM), a semi-log plot (percentrelative transport of oratate vs. time) was generated to determine theconcentration at which 50% inhibition was observed (i.e., the IC50).

The results of these assays for compounds according to Formula (I) areshown in Table 1:

TABLE 1 URAT1 Xanthine Oxidase Compound IC50 (μM) IC50 (μM) Formula(I_(a)) >4.8 0.25 Formula (I_(b)) 54.1 Formula (I_(c)) >150 1.2 Formula(I_(d)) >300 Formula (I_(e)) >37.5 1.6 Formula (I_(f)) >9 0.20 Formula(I_(g)) >150 0.55 Formula (I_(h)) >18.8 0.74 Formula (I_(i)) >295.8Formula (I_(j)) 13.1 Formula (I_(k)) 1.3 Formula (I_(l)) 6.3 Formula(I_(m)) 18.2 Formula (I_(n)) 292 Allopurinol >300^(†) 2.0 to 5.0Lesinurad 18.61* >300^(†) 52.5 ± 539^(†)* ^(†)Presentation estimate;Proc. EULAR Abstract #THU0357, 2008 *URAT1 assay as described herein

The results of these assays for compounds according to Formula (II) areshown in Table 2:

TABLE 2 URAT1 Xanthine Oxidase Compound IC50 (μM) IC50 (μM) Formula(II_(a)) 9.2 Formula (II_(b)) 12.1 0.56 Formula (II_(c)) 2.4 0.05Formula (II_(d)) 1.07 ≤0.02 Formula (II_(e)) >9.45 0.025 Formula(II_(f)) 217 Formula (II_(g)) 2.8 Formula (II_(h)) 1.8 Allopurinol>300^(†) 2.0 to 5.0 Lesinurad 18.61* >300^(†) 52.5 ± 5.9^(†)*

The results of these assays for compounds according to Formula (III) areshown in Table 3:

TABLE 3 URAT1 Xanthine Oxidase Compound IC50 (μM) IC50 (μM) Formula(III_(a)) 4.5 Allopurinol >300^(†) 2.0 to 5.0 Lesinurad 18.61* >300^(†)52.5 ± 5.9^(†)*

The results of these assays for compounds according to Formula (IV) areshown in Table 4:

TABLE 4 URAT1 Xanthine Oxidase Compound IC50 (μM) IC50 (μM) Formula(IV_(a)) 8.6 Formula (IV_(b)) >300 Formula (IV_(c)) >300 Allopurinol>300^(†) 2.0 to 5.0 Lesinurad 18.61* >300^(†) 52.5 ± 5.9^(†)*

The results of these assays for compounds according to Formula (V) areshown in Table 5:

TABLE 5 URAT1 Xanthine Oxidase Compound IC50 (μM) IC50 (μM) Formula(V_(a)) 36.1 Allopurinol >300^(†) 2.0 to 5.0 Lesinurad 18.61* >300^(†)52.5 ± 5.9^(†)*

The results of these assays for compounds according to Formula (VI) areshown in Table 6:

TABLE 6 URAT1 Xanthine Oxidase Compound IC50 (μM) IC50 (μM) Formula(VI_(a)) >37.5 0.61 Formula (VI_(b)) >27 0.14 Formula (VI_(c)) 2.5Allopurinol >300^(†) 2.0 to 5.0 Lesinurad 18.61* >300^(†) 52.5 ±5.9^(†)*

The results of these assays for compounds according to Formula (VII) areshown in Table 7:

TABLE 7 URAT1 Xanthine Oxidase Compound IC50 (μM) IC50 (μM) Formula(VII_(a)) 7 Allopurinol >300^(†) 2.0 to 5.0 Lesinurad 18.61* >300^(†)52.5 ± 5.9^(†)*

The results of these assays for compounds according to Formula (VIII)are shown in Table 8:

TABLE 8 URAT1 Xanthine Oxidase Compound IC50 (μM) IC50 (μM) Formula(VIII_(a)) 17.4 Formula (VIII_(b)) 73.8 Allopurinol >300^(†) 2.0 to 5.0Lesinurad 18.61* >300^(†) 52.5 ± 5.9^(†)*

All of the compounds tested inhibited at least one of URAT1 and xanthineoxidase. Formulae (I_(a)), (I_(c)), (J_(e)), (I_(f)), (I_(g)), (I_(h)),(I_(k)), (II_(b)), (II_(d)), (II_(e)), (VI_(a)) and (VI_(b)) areparticularly potent inhibitors of xanthine oxidase compared toallopurinol. Several of the compounds also effectively inhibit URAT1,although not all of them were tested. These are bifunctional inhibitors.One representative example of a particularly effective bifunctionalinhibitor is Formula (II_(d)).

While many compounds were potent inhibitors, the extent of inhibition ofeach enzyme/channel was different. Such variability allows theintelligent selection of a pharmaceutically acceptable product thatexhibits greater or lesser inhibition of one or the other enzyme target.For example, greater inhibition of XO might be deemed preferable for apatient whose primary metabolic defect was over-production of uric acid.Conversely, greater inhibition of URAT1 might be deemed preferable for apatient whose primary metabolic defect was under-excretion of uric acid.However, it should be noted that almost all patients with hyperuricemiawill benefit from reduction in serum uric acid, and bifunctionalcompounds can be expected to exert a beneficial effect in such patients.The practitioner, guided by the present disclosure, will be able toselect particular compounds as appropriate for a specific use based onthe level of skill in the art.

By way of comparison, allopurinol has an IC50 for XO ranging from about2.0 to about 5.0 μM and an IC50 for URAT1 of >300 μM. Lesinurad has anIC50 for XO of >300 μM and an IC50 for URAT1 ranging from 18 to 53 μM.Thus, neither of these compounds is considered bifunctional, since bothare selective inhibitors of only one enzyme that affects eitherproduction or excretion of uric acid. In contrast, certain of thecompounds described herein are not only bifunctional, several aresubstantially more potent inhibitors of either or both XO and URAT1.

While in many clinical situations it is desirable to treat hyperuricemiawith a drug that is highly potent against both XO and URAT1, it is alsocontemplated that selection of a particular compound of the inventionfor treatment of hyperuricemia may be based on the phenotype of thehyperuricemic patient being treated (i.e., the relative contributions ofover-production of uric acid and under-excretion of uric acid to thepatient's specific disease). Where over-production of uric acidpredominates, use of compounds according to the invention that aresubstantially more potent against XO than URAT1 may be appropriate.Where under-excretion of uric acid predominates, use of compoundsaccording to the invention that are substantially more potent againstURAT1 than XO may be appropriate. Although the genetics of these twopathways are not completely understood, chemical testing to determinethe extent to which each contributes to the hyperuricemia of aparticular patient has been published, and is expected to be useful todetermine the patient's disease phenotype for selection of anappropriate drug.

Although the invention herein has been described with reference toparticular embodiments, it is to be understood that these embodimentsare merely illustrative of the principles and applications of thepresent invention. It will be apparent to those skilled in the art thatvarious modifications and variations can be made to the method andapparatus of the present invention without departing from the spirit andscope of the invention. Thus, it is intended that the present inventioninclude modifications and variations that are within the scope of theappended claims and their equivalents.

What is claimed is:
 1. A compound selected from the group consisting ofa) compounds having a structure represented by Formula (I):

wherein W is selected from O, S, and NR², Y is selected from OH andN(R²)₂, X is selected from O, S, and NR²; T is —CONR²—, —C(NR²)NH—,—C(NOR²)NH—, —C(N—NR²)NH—, —C(SR²)N—, or —NHC(O)—; A is phenyl,heteroaryl, C5-C10 branched or unbranched cycloalkyl, C6-C10bicycloalkyl or C5-C10 spirocycloalkyl; each Z is independently presentor absent and, if present, is independently selected from one or morehalogen atoms, —CN, —CF₃, —OR², —C(O)R², SR², —S(O)_(g)R³ where g is 1or 2, —N(R²)₂, —NO₂, —CO₂R², —OCO₂R³, OC(O)R², —CON(R²)₂, —NR²C(O)R²,—SO₂N(R²)₂, —NR²SO₂R³, —NR²SO₂N(R²)₂ or —NR²C(O)N(R²)₂, —C(O)NHOR²,alkyl, aryl, alkenyl and alkynyl; wherein each R² is independently H,alkyl or aryl; wherein each R³ is independently alkyl or aryl,optionally substituted with one or more halogen atoms or OR²; andwherein a, b, c, d, and e are each independently carbon or nitrogen, orfour of a, b, c, d, and e are each independently carbon or nitrogen andone of a, b, c, d, and e is O, with the proviso that at least one of a,b, c, d and e is nitrogen, and Z is not connected directly to nitrogenor oxygen; and tautomers thereof, b) compounds having a structurerepresented by Formula (III):

wherein W is selected from O, S, and NR², Y is selected from OH andN(R²)₂, X is selected from O, S, and NR²; each R² is independently H,alkyl or aryl; and f is divalent —CR²—, —C(O)—, —SR², —S(O)_(g)— where gis 1 or 2, —N(R′)₂—; or —C(—O(CR²)_(n)O—)— where n=2-3; and tautomersthereof, c) compounds having a structure represented by Formula (IV):

wherein W is selected from O, S, and NR², Y is selected from OH andN(R²)₂, X is selected from O, S, and NR²; each R² is independently H,alkyl or aryl; and f is divalent —CR²—, —C(O)—, —S(O)_(g)— where g is1-2, —N(R²)₂—; or —C(—O(CR²)_(n)o-)- where n is 2-3; and tautomersthereof, d) compounds having a structure represented by Formula (V):

wherein W is selected from O, S, and NR², Y is selected from OH andN(R²)₂, X is selected from O, S, and NR²; and each R² is independentlyH, alkyl or aryl; and tautomers thereof, e) compounds having a structurerepresented by Formula (VI):

wherein W and X are each independently O, S, NR² or N(R²)₂; A is phenyl,heteroaryl, C5-C10 branched or unbranched cycloalkyl, C6-C10bicycloalkyl or C5-C10 spirocycloalkyl; each Z is independently presentor absent and, if present, is independently selected from one or morehalogen atoms, —CN, —CF₃, —OR², —C(O)R², SR², —S(O)_(g)R³ where g is 1or 2, —N(R²)₂, —NO₂, —CO₂R², —OCO₂R³, OC(O)R², —CON(R²)₂, —NR²C(O)R²,—SO₂N(R²)₂, —NR²SO₂R³, —NR²SO₂N(R²)₂ or —NR²C(O)N(R²)₂, —C(O)NHOR²,alkyl, aryl, alkenyl and alkynyl; U is —O—, —S—, —NR²— or —S(O)_(g)—where g is 1 or 2; wherein each R² is independently H, alkyl or aryl;wherein each R³ is independently alkyl or aryl, optionally substitutedwith one or more halogen atoms or OR²; and wherein a, b, c, d, and e areeach independently carbon or nitrogen, or four of a, b, c, d, and e areeach independently carbon or nitrogen and one of a, b, c, d, and e is O,with the proviso that at least one of a, b, c, d and e is nitrogen, andZ is not connected directly to nitrogen or oxygen; and tautomersthereof, f) compounds having a structure represented by Formula (VII):

wherein X and Y are each independently O, S, NR² or N(R²)₂; Z is presentor absent and, if present, is selected from one or more halogen atoms,—CN, —CF₃, —OR², —C(O)R², SR², —S(O)_(g)R³ where g is 1 or 2, —N(R²)₂,—NO₂, —CO₂R², —OCO₂R³, OC(O)R², —CON(R²)₂, —NR²C(O)R², —SO₂N(R²)₂,—NR²SO₂R³, —NR²SO₂N(R²)₂ or —NR²C(O)N(R²)₂, —C(O)NHOR², alkyl, aryl,alkenyl and alkynyl, wherein each R² is independently H, alkyl or aryl;wherein each R³ is independently alkyl or aryl, optionally substitutedwith one or more halogen atoms or OR²; and wherein a, b, c, d, and e areeach independently carbon or nitrogen, or four of a, b, c, d, and e areeach independently carbon or nitrogen and one of a, b, c, d, and e is O,with the proviso that at least one of a, b, c, d and e is nitrogen, andZ is not connected directly to nitrogen or oxygen; and tautomersthereof, and g) compounds having a structure represented by Formula(VIII):

wherein W, X, and Y are each independently O, S, NR² or N(R²)₂; T is—CONR²—, —C(NR²)NH—, —C(NOR²)NH—, —C(N—NR²)NH—, —C(SR²)N—, or —NHC(O)—;A is phenyl, heteroaryl, C5-C10 branched or unbranched cycloalkyl,C6-C10 bicycloalkyl or C5-C10 spirocycloalkyl; each Z is independentlypresent or absent and, if present, is independently selected from one ormore halogen atoms, —CN, —CF₃, —OR², —C(O)R², SR², —S(O)_(g)R³ where gis 1 or 2, —N(R²)₂, —NO₂, —CO₂R², —OCO₂R³, OC(O)R², —CON(R²)₂,—NR²C(O)R², —SO₂N(R²)₂, —NR²SO₂R³, —NR²SO₂N(R²)₂ or —NR²C(O)N(R²)₂,—C(O)NHOR², alkyl, aryl, alkenyl and alkynyl; wherein each R² isindependently H, alkyl or aryl; wherein each R³ is independently alkylor aryl, optionally substituted with one or more halogen atoms or OR²;and tautomers thereof.
 2. The compound according to claim 1, wherein the5-member heterocyclic ring is a substituted or unsubstituted triazole.3. The compound according to claim 1, wherein-XR¹ is —SCH₃ or —OCH₃. 4.The compound according to claim 1 wherein, in Formula (I): a) X and Ware each independently O or S; b) Y is OH or NH₂; c) T is —C(NH)NH—,—NHC(O)—, —C(SCH₃)N—, —C(NOH)NH—, —C(N—NH₂)NH—, or —CONH; d) each R² onthe barbiturate ring is independently H or CH₃; e) A is phenyl, branchedcycloalkyl, or unbranched cycloalkyl; and f) Z, if present, is phenyl orCF₃.
 5. The compound according to claim 1, wherein, in Formula (III), fis —S(O)₂—.
 6. The compound according to claim 1 wherein, in Formula(IV), f is —NH—, —C(O)— or —C(—O(CH₂)₂O—)—.
 7. The compound according toclaim 6, wherein X on the fused ring structure is O.
 8. The compoundaccording to claim 1 wherein, in Formula (V), each R² is H.
 9. Thecompound according to claim 1 wherein, in Formula (VI), U is —O— or—NH—, and the 5-member heterocyclic ring is triazole.
 10. The compoundaccording to claim 1 wherein, in Formula (VII), the unfused 5-memberheterocyclic ring is triazole.
 11. A pharmaceutical compositioncomprising a compound having a structure represented by any of Formulae(I), and (III)-(VIII); a tautomer of any of Formulae (I), and(III)-(VIII), or a combination thereof, and a pharmaceuticallyacceptable carrier.
 12. The pharmaceutical composition according toclaim 11, which is formulated for controlled or extended release of thecompound or combination thereof.
 13. The pharmaceutical compositionaccording to claim 11, wherein the pharmaceutically acceptable carrieris selected from the group consisting of water or saline, a solvent, adispersing agent, a coating, a surfactant, a preservative, an emulsion,an alcohol, a polyol, and an isotonic agent.
 14. A method for reducinguric acid levels in blood or serum of a subject, or preventing elevationof uric acid levels in blood or serum of a subject, comprisingadministering to a subject in need thereof a compound having a structurerepresented by any of Formulae (I), and (III)-(VIII)); a tautomer of anyof Formulae (I), and (III)-(VIII), or a combination thereof, in anamount effective to reduce blood or serum uric acid levels.
 15. Themethod according to claim 14, wherein administering the compound treatsor prevents a disorder of uric acid metabolism caused by, or associatedwith, hyperuricemia.
 16. The method of claim 14, wherein the disorder ofuric acid metabolism is selected from the group consisting of gout,hyperuricemia, tumor lysis syndrome, kidney disease, arthritis, kidneystones, kidney failure, urolithiasis, plumbism, hyperparathyroidism,psoriasis, inborn genetic errors of metabolism, Lesch-Nyhan syndrome,sarcoidosis, cardiovascular disease, atherosclerosis, and disorders ofuric acid metabolism associated with transplantation of blood, bonemarrow or solid organs.
 17. The method according to claim 14, wherein adaily dose of about 20 to about 1,500 mg/m²/day is administered.
 18. Themethod according to claim 14, wherein the compound or combinationthereof is administered by injection, infusion, or oral administration.19. The method according to claim 18, wherein the compound orcombination thereof is administered by intravenous infusion or bolusinjection.